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A Hitchhiker’s Guide to Supplying Enzymatic Reducing Power into Synthetic Cells

Cite this: ACS Synth. Biol. 2023, 12, 4, 947–962
Publication Date (Web):April 13, 2023
https://doi.org/10.1021/acssynbio.3c00070

Copyright © 2023 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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The construction from scratch of synthetic cells by assembling molecular building blocks is unquestionably an ambitious goal from a scientific and technological point of view. To realize functional life-like systems, minimal enzymatic modules are required to sustain the processes underlying the out-of-equilibrium thermodynamic status hallmarking life, including the essential supply of energy in the form of electrons. The nicotinamide cofactors NAD(H) and NADP(H) are the main electron carriers fueling reductive redox reactions of the metabolic network of living cells. One way to ensure the continuous availability of reduced nicotinamide cofactors in a synthetic cell is to build a minimal enzymatic module that can oxidize an external electron donor and reduce NAD(P)+. In the diverse world of metabolism there is a plethora of potential electron donors and enzymes known from living organisms to provide reducing power to NAD(P)+ coenzymes. This perspective proposes guidelines to enable the reduction of nicotinamide cofactors enclosed in phospholipid vesicles, while avoiding high burdens of or cross-talk with other encapsulated metabolic modules. By determining key requirements, such as the feasibility of the reaction and transport of the electron donor into the cell-like compartment, we select a shortlist of potentially suitable electron donors. We review the most convenient proteins for the use of these reducing agents, highlighting their main biochemical and structural features. Noting that specificity toward either NAD(H) or NADP(H) imposes a limitation common to most of the analyzed enzymes, we discuss the need for specific enzymes─transhydrogenases─to overcome this potential bottleneck.

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 Special Issue

Published as part of the ACS Synthetic Biologyvirtual special issue “Synthetic Cells”.

Introduction

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Since the early 1990s, when in vitro physicochemical conditions leading to synthetic self-replicating lipid vesicles (liposomes) were initially explored, (1,2) efforts toward the construction of artificial cells by rationally putting together well-defined (bio)chemicals have been intensified. The encapsulation into liposomes of more sophisticated biomolecular mixtures, such as the translational machinery, (3,4) set a next milestone to recreate “bottom-up” biomimetic systems with greater functional complexity. In recent years, the growing focus on synthetic cells assembled by a bottom-up approach led to the exciting development of new subfields within a broader research area defined as synthetic biology. (5−9) One of these fields covers the design of enzymatic pathways/modules able to perform metabolic functions typical of living systems within the compartment of synthetic liposomes. Central energy converting modules have been successfully reproduced inside liposomes, including the production of ATP, (10−12) the generation of light-driven proton motive force, (13) and the reduction of NAD(P)+. (14) Nonetheless, the delineation of an essential metabolism that guarantees physicochemical homeostasis, growth, and replication still remains a daunting task, because the metabolic modules must operate in a coordinated way. Therefore, early design choices of a synthetic metabolism are crucial to allow for expansion and combination of modules. The amount of possible choices is large, because life has found different solutions to thrive in the most disparate environments and variable access to nutrients thanks to a wide range of genes coding for enzymes that allow it to harness the available resources. (15,16)
Despite the enormous diversity of metabolic pathways, there are a few molecules (hub metabolites) taking part in hundreds of different reactions in all the living systems. (17,18) Specifically, the nicotinamide adenine dinucleotides NAD(H) and NADP(H) are involved in approximately 2000 out of almost 8000 reactions annotated in the Escherichia coli Metabolome Database (19) (Figure 1), and therefore designated as hub metabolites. Their main function is to transfer reducing equivalents to or from a plethora of reactants, via the catalytic action of specific oxidoreductases. (20) The existence of two nicotinamide cofactors that differ only in structure at the position 2′ of the riboside ring where NAD(H) has a hydroxyl group, which in NADP(H) is phosphorylated, allows for the distinction of pathways that break down (catabolism) or assemble (anabolism) molecular building blocks. (21) NADPH is mostly involved in anabolic reduction reactions and NAD+ in catabolic oxidations. For instance, the cellular NADPH concentration in most bacteria is usually kept much larger than the NADP+ concentration, while the NAD+/NADH ratio is lower, thus enabling oxidations. (22) However, this dichotomy is not a strict rule for all living systems, and the use of NADP(H) or NAD(H) in specific reactions is dependent on enzyme specificities, thermodynamic constraints, and cellular conditions. (22−24) The continuous reduction and reoxidation of the nicotinamide pools is necessary to feed further enzymatic reactions involved in the biosynthesis of crucial biomolecules such as sugars, nucleotides, lipids, and cofactors. (20,22,25,26)

Figure 1

Figure 1. NAD(H) and NADP(H) occurrence among the enzymatic reactions of Escherichia coli. Metabolites involved in cellular reactions tend to follow a power-law distribution (generically described by the equation y = C/xa), in which the majority of the metabolites is involved in a small number of reactions while a small group of metabolites takes part in up to 100 enzyme-mediated reactions. On the left y-axis is reported the absolute number of metabolites (n), and on the right y-axis is shown the same number of metabolites in terms of percentage (%). The diagonal approximates the linearity of the power-law distribution in a double logarithmic scale graph, as also shown by Schmidt et al. (17) The restricted group of metabolites involved in more than a hundred reactions represent exceptions to the power-law distribution, and are called hub metabolites. The upper right inset highlights the number of reactions dependent on NAD+ (red diamond), NADH (red-black diamond), NADP+ (blue diamond), and NADPH (blue-black diamond). The data set was extracted from the Escherichia coli Metabolome Database (http://www.ecmdb.ca).

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Several reviews have discussed the enzymatic regeneration of NAD((P)H) cofactors both in vivo and in vitro, principally for (bio)manufacturing purposes. (22,27−30) However, building a redox regeneration pathway that supports a minimal metabolic network enclosed in liposome confinement and built in a bottom-up manner poses specific challenges, which we will address in this work. We review (i) the choice of the suitable electron donor (substrate) for reduction of nicotinamides from a thermodynamic perspective, (ii) transport modes of the potential electron donating substrates across the phospholipid bilayer, (iii) the availability of NAD(P)+-dependent enzymes that use the substrate, (iv) the fate of the reaction product formed during the nicotinamide reduction, which preferentially should not accumulate in the lumen of the synthetic cells, and (v) the compatibility of the redox power provision with an aerobic environment, considering the integration of the redox module in a more complex metabolic network that might benefit from the presence of oxygen. These apparently simple conditions narrow the number of compounds to take into account as convenient electron donors, and enzymes to be implemented for utilization of the reducing power. We focus here on electron supply by chemical donor molecules rather than photoinducible water-splitting systems (e.g., photosystems I and II). (31) While photosynthetic reactions are intriguing options to regenerate NAD(P)H, the reconstitution of the full photosystem and accessory proteins to achieve a functional redox module demands a large number of proteins and coenzymes. Additionally, this would tie the electron supply to light exposure of the synthetic cell for operation.
Finally, we discuss enzymatic transhydrogenation, which is of relevance to synthetic cells because dehydrogenases oxidizing the respective electron donating substrate usually exhibit cofactor specificity for either NAD(H) or NADP(H), whereas a synthetic metabolism would require both the distinct cofactors in certain ratios for energy production and biosynthetic purposes, respectively.

The Rationale for Selecting Electron Donors for Nicotinamide Cofactors in Synthetic Cells

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1. The Thermodynamics

Thermodynamic feasibility is an essential factor in the design of metabolic pathways: cell-free synthetic pathways must rely on initial thermodynamic analysis to ensure the desired directionality of metabolic fluxes. (37,38) When constructing synthetic cells, the same principle must be applied in linking the different pathways for synergic functionality of the overall enzymatic network. For redox reactions, the spontaneity of a reaction under biological standard conditions (ΔG°′ < 0, pH 7.0, 25 °C, 1 atm) can be calculated from the difference in reduction potentials (ΔE°′) between the reactants, according to eq 1:
Δ G ° ′ = − n F Δ E ° ′
(1)
where ΔG°′ stands for the change in Gibbs free energy under biological standard conditions, n corresponds to the number of electrons involved in the reaction, and F is the Faraday constant. Since the electrons spontaneously move in the direction of the redox pair with the most positive E°′ value, the hydride transfer from a reduced to an oxidized species is constrained by the reduction potentials of the involved reactants. A backward electron flow can occur by shifting the relative concentrations of reactants, but it may require substantial concentration differences to overcome an unfavorable equilibrium. (39) Taking pH 7.0 for a synthetic cell to operate, the standard reduction potential value E°′ of the half-reaction NAD(P)+/NAD(P)H at −0.32 V limits the number of potential electron donors. With their lower E°′ values (Figure 2), the phosphite/phosphate, sulfite/sulfate, carbon monoxide (CO)/CO2, d-glucose/CO2, formic acid/CO2, molecular hydrogen (H2)/H+, and isocitrate/α-ketoglutarate redox couples emerge as thermodynamically favorable “reducing agents” for nicotinamides.

Figure 2

Figure 2. Overview of the standard reduction potentials (E°′, at 25 °C, pH 7.0) of the half-reactions of candidate electron donors. (32−36)

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The malate/oxaloacetate pair (with E°′ value of −0.17 V) represents a less favorable option from a thermodynamic perspective. Under standard conditions, the electrons would move from NAD(P)H to oxaloacetate, with an equilibrium constant strongly shifted toward the formation of NAD+ and malate (Keq ≈ 3 × 105). (40) However, with elevated concentrations of malate the reaction can also lead to reduction of NAD(P)+, as it happens in vivo in the tricarboxylic acid cycle. (41)
The succinate/fumarate redox pair has an E°′ value (−0.04 V) even further above NAD(P)H/NAD(P)+ making it virtually impossible to reduce NAD(P)+ with electrons from succinate. In nature also no NAD(P) +-dependent succinate dehydrogenase is known, and in living cells this reaction operates with quinones as (high potential) electron acceptors.

2. The Accessibility to a Membrane-Defined Compartment Characterized by Selective Permeability

In a cell-mimicking system, the phospholipid membrane delineates a selectively open system. (42) The NAD(P)+ cofactors and the enzymes remain confined within the lumen of the liposomal compartment once encapsulated due to their membrane-impermeable nature. Only small uncharged molecules can pass through the membrane unassisted, while charged and larger molecules require specific membrane proteins to allow the transport of solutes. (33) While pore-forming proteins (e.g., cytolysin A, α-hemolysin, fragaceatoxin C, OmpF, etc.) (43) can be employed to enable the passage of multiple reactants within a certain size depending on the cutoff of the specific pore, most membrane transporters are characterized by substrate-specificity (44) that ensures a high degree of selectivity and control of membrane-impermeable reactants entering or leaving the liposomal lumen. Although pore-forming proteins have found some useful applications in cell-like systems, (45−47) they are not ideal for sustaining the out of equilibrium status over the long-term, hindering the retention and internal recycling of the small molecules necessary for metabolic homeostasis. (42) We therefore refer specifically to transporters as membrane proteins of choice in bottom-up synthetic cells to mediate the translocation of membrane-impermeable solutes.
When an external electron donating substrate is membrane-impermeable, the addition to the system of a transporter is needed in order to ensure the supply of substrate to the luminal dehydrogenase: this is the case when utilizing electron donors such as phosphite, malate, isocitrate, and d-glucose. In contrast, small molecules such as H2, formic acid and glycerol can permeate across the membrane by unassisted diffusion. For formate, at physiological pH, the anionic form (HCOO–) prevails over the protonated formic acid (pKa = 3.75). Only the latter species is membrane permeable but the acid–base equilibrium does not limit diffusion through phospholipid membranes: its permeability coefficient has been estimated at 719 × 10–5 cm/s in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles. (48) Although almost 2 orders of magnitude less permeable than formic acid, (48) glycerol is also membrane-permeable and consequently does not strictly require a specialized transporter. Glycerol has being recognized in recent years as an attractive hydrogen donor for the synthesis of value-added chemicals. (49,50)
Finally, the electrochemical gradient resulting from the import of an electron donor must be taken into consideration because it may affect the pH gradient across the membrane, cause osmotic changes, and may lead to variations to the electrical potential difference across the membrane, if net charge translocation takes place, which could alter the functionality of the synthetic cell. (51) Potentially, a careful design of the phenomena that take place at the level of the membrane enclosing the compartment (e.g., proton gradient, membrane potential) can be exploited for other essential processes, such as energy production (52) or cell division. (53) The same reasoning applies to products that diffuse out passively or that are exported by protein-mediated action.

3. The Availability of Annotated NAD(P)+-Dependent Dehydrogenases

A low reduction potential and accessibility to the lumen alone are not sufficient to select suitable hydride donors for NAD(P)+ coenzymes. There must also be oxidoreductases mediating the oxidation of the electron donor in favor of the specific reduction of the nicotinamide cofactor. The absence of a catalyst would make the time scale of the reaction incompatible with life-like systems. (54) Two clear examples of such limitation are represented by the redox couples sulfite/sulfate and carbon dioxide/carbon monoxide. Although SO42–/HSO32– has a much more negative reduction potential (E°′ = −0.52 V) (32) than the nicotinamide couples, there is not a single known enzyme that transfers reducing equivalents directly from sulfite to NAD+ or NADP+. Sulfite can be oxidized enzymatically only by reducing either ferricytochrome c by sulfite dehydrogenase (EC 1.8.2.1) in bacteria or oxygen by the action of sulfite oxidase (EC 1.8.3.1) in plants and animals. (55) Similarly, CO would constitute a suitable electron donor for nicotinamides from a thermodynamic point of view (CO2/CO, E°′ = −0.52 V), yet carbon monoxide dehydrogenases (CODHs) mostly use oxidized ferredoxin (EC 1.2.7.4) (56) or quinones (EC 1.2.5.3) (57) as reaction partners. To date, the oxygen-tolerant CODH from the hyperthermophilic archaeon Aeropyrum pernix has been the only purified homologue reported to reduce NAD(P)+ cofactors. (58)
Contrary to sulfite and carbon monoxide, glycerol can be oxidized by the action of specific NAD+-dependent glycerol dehydrogenases (EC 1.1.1.6) (59) forming dihydroxyacetone and NADH. The thermodynamic bottleneck between the redox couples dihydroxyacetone/glycerol (E°′ = −0.24 V) (36) and NAD(P)+/NAD(P)H is smaller than what was mentioned above for the dicarboxylates, and therefore is overcome by relatively minor changes in concentration of the reactants. These factors led us to consider glycerol as a suitable electron donating substrate for nicotinamide cofactors.
For the later selected electron donors, NAD(P)+-dependent dehydrogenases are available as extensively discussed below.

4. NAD((P)H) Regeneration Systems without Undesired or Reaction Products

Synthetic cells must perform a diverse set of biochemical reactions to successfully maintain their homeostasis and eventually be able to reproduce themselves. Ideally, any new reactant supplied from the outside has to fulfill specific functions by undergoing a catalytic conversion that results in the formation of new building blocks and/or enables the luminal regeneration of needed biomolecules. Therefore, when the reaction products have no use and can be even detrimental to the metabolic network, it is necessary to remove them from the liposome compartment to avoid accumulation preventing any possible interference with other metabolic components. To efficiently ensure the removal of a specific membrane-impermeable product, it may be possible to take advantage of a membrane protein antiporter exporting the undesired reaction product in exchange with an external substrate of interest, as showcased in liposomes encapsulating the arginine breakdown pathway for ATP production: (10) in this case, the product ornithine is exported out in exchange with arginine, that is the initial substrate required to trigger the pathway activity. With regard to redox reactions, the best electron donors are the compounds whose oxidation transfers hydrides to NAD(P)+ cofactors while the coproducts can be easily exported out (Figure 3). This criterion of “orthogonality” leads to the exclusion of some of the above-mentioned compounds that thermodynamically are very advantageous with their low E°′ value, for instance d-glucose and α-ketoglutarate, as their direct oxidation products, β-d-glucono-1,5-lactone and alpha-ketoglutarate, cannot be easily exported. Even though both molecules could partly serve as building blocks, this would be challenging to stoichiometrically balance with the need for reducing equivalents in a synthetic cell, and likely still some transport mechanisms would be needed to export a part of this product pool.

Figure 3

Figure 3. Strategies for the orthogonal provision of reducing power in synthetic cells. Both the electron donors for the nicotinamide coenzymes and the respective reaction products have two possible ways to move across the membrane: by protein-mediated action (A) or by unassisted permeation (B–D). (A) Phosphite is internalized by an ATP-binding cassette (ABC) transporter (shown in green), while its reaction product phosphate can be exported in symport with a proton (in yellow) or a sodium ion (in orange), or by facilitated-diffusion (in red). (B–D) Formate, molecular hydrogen and glycerol, as well as carbon dioxide and dihydroxyacetone freely and readily permeate the phospholipid bilayer. In order to transfer the hydrides from the donor to the NAD(P)+ cofactors, the cell-like compartments need to be equipped with specific dehydrogenase(s).

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Another potential electron donor substrate is methanol, however its oxidation product formaldehyde is highly toxic and known to cross-link proteins and DNA and hence is an undesired option. (60)
To further elaborate on the export issue for d-glucose products, this sugar can, apart from single-step oxidation to β-d-glucono-1,5-lactone, also be fully oxidized to the easy-to-export product CO2. In living systems the full oxidation is performed using multienzymatic metabolic pathways (Emden–Meyerhof or Entner–Doudoroff glycolysis in combination with the citric acid cycle). (61) These multistep pathways are obviously extremely advantageous in vivo, but they are challenging to use in the context of a synthetic cell, as they would require a considerable number of enzymes to be purified and reconstituted, which may lead to technical complications as well as potential regulatory complexity. In addition, the tricarboxylic acid cycle not only generates NAD(P)H but also reduced quinol, which is not necessarily desired and requires another regeneration system such as a respiratory chain, further increasing complexity and hindering orthogonality of the redox regeneration system.
As an alternative for the glycolytic routes using d-glucose, a single enzyme such as d-glucose dehydrogenase (GDH, EC 1.1.1.47) could be employed to directly form NAD(P)H while oxidizing the hexose carbohydrate into β-d-glucono-1,5-lactone. Nonetheless, export of β-d-glucono-1,5-lactone is not known to occur via an antiporter with glucose, nor any other form of transport or minimal metabolic route for disposal is known for this product, and therefore accumulation of the metabolite would occur.
The same applies for the isocitrate/α-ketoglutarate couple, unless a specific carboxylate antiporter can be identified and successfully utilized in the desired direction (uptake of isocitrate and export α-ketoglutarate), which would allow to retain only the reducing equivalents, as NAD(P)H, in the lumen of synthetic cells enclosing an isocitrate dehydrogenase (EC 1.1.1.41 and 1.1.1.42), since the carbon dioxide produced together with α-ketoglutarate would diffuse out.

5. The Preference for Aerobic Pathways by Envisioning Metabolic Complexity

Some enzymes catalyzing redox reactions are oxygen-sensitive, and only functional in anaerobic conditions (e.g., many oxidoreductases and respiratory enzymes from anaerobic microorganisms (62−64)) forcing the assembly and function of the vesicles to an inconvenient oxygen-free environment. Indeed, it is technically challenging and practically undesirable to prevent the presence of oxygen in each stage of the liposomal preparation, starting from the biochemical encapsulation to the functional assays of vesicle activity, regardless of the chosen technique or the size of the phospholipid compartments. (65) On top of this technical limitation, it may not be desirable to exclude molecular oxygen, as it drives one of the most efficient energy conversion processes as a strong oxidizing agent, that is the generation of a proton motive force across the membrane, which subsequently is used for the synthesis of ATP. (66,67) Considering the high demand for ATP calculated to support the major functions in a synthetic cell, (11) the option of a minimal respiratory system with oxygen as final electron acceptor is ultimately to be considered: it has been estimated that aerobic respiration in microbes leads to an ATP yield per electron that is 1–2 orders of magnitude higher than alternative anaerobic processes (e.g., denitrification, sulfate and ferric respiration, methanogenesis). (68) In this respect, the use of complex I concomitantly with ubiquinone and an alternative oxidase has been proved as a promising strategy in (proteo)liposomes to fuel a minimal respiratory chain with oxygen as a final electron sink. (69) Its recent coupling with an ATP-synthase (70) has even shown the potential of generating ATP by taking advantage of the proton motive force established by the proton-pumping action of the mitochondrial complex I; however, in this case ATP was produced outside the vesicles as a result of the preferential orientation of complex I.
Furthermore, artificial cells capable of transcription and translation (TX-TL) are often tested and optimized with green fluorescent proteins and derivatives to monitor the protein synthesis efficiency. Folding and maturation of these fluorescent proteins is strictly dependent on dioxygen availability. (3) Hence, the enzymes further discussed in this review are oxygen tolerant.

The Protein Toolbox for Redox Reactions Inside Synthetic Cells

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The aforementioned considerations led us to identify four main candidate compounds as viable electron donors for nicotinamides within artificial cells (Table 1): phosphite, formate, molecular hydrogen, and glycerol. We now proceed to describe the most efficient ways exploit their reducing power (Figure 3) by focusing on the enzymes that oxidize them (dehydrogenases listed in Table 2) and, in the case of phosphite/phosphate, allow their transport through the lipid bilayer (membrane transporters). The other donors and products can passively enter and leave the cell.
Table 1. Properties of the Electron Donating Substrates and Respective Reaction Products Selected as Suitable for Synthetic Cellsa
compound PubChem ID MW standard reduction potential (V) solubility transport mode P × 10–5 (cm/s)
Phosphite (HPO32–) 107908 78.97   high primary active impermeable
Phosphate (PO43–) 1061 94.97 –0.65 high secondary active impermeable
facilitated diffusion
Formate (HCOO–) 283 45.02   high passive diffusion 719
Carbon dioxide (CO2) 280 44.01 –0.43 low passive diffusion 1 × 105
Hydrogen (H2) 783 2.02   low passive diffusion N.D.
Hydrogen ion (H+) 1038 1.01 –0.41 high primary active 1 × 10–9
secondary active
Glycerol (C3H8O3) 753 92.09   high passive diffusion 2
Dihydroxyacetone (C3H8O3) 670 90.08 –0.24 high passive diffusion N.D.
a

The reported permeability coefficients (P) refer to (DOPC) vesicles. (48,71) N.D. stands for “not determined” in an experimental setup.

Table 2. Features of the NAD(P)+-Dependent Dehydrogenases Oxidizing Suitable Electron Donors for Nicotinamide Coenzymes
enzyme name enzyme code organism substrates KM (mM) kcat (s–1) molar mass (kDa) oligomeric state pH optimum temp. optimum (°C) ref.
Phosphite dehydrogenase 1.20.1.1 P. stutzeri NAD+ 0.05 3.2 70 homodimer 7.0–8.0 35 (34,72)
phosphite 0.05
Formate dehydrogenase 1.17.1.9 P. species 101 NAD+ 0.08 7.5 90 homodimer 6.0–9.0 63 (73,74)
formate 15.0
Soluble [NiFe]-hydrogenase 1.12.1.2 C. necator NAD+ 0.20 109.0 170 heterotetramer 8.0 35 (75−77)
H2 0.04 143.0 210 heterohexamer
Glycerol dehydrogenase 1.1.1.6 G. stearothermophilus NAD+ 0.52 7.4 320 homo octamer 9.0–10.0 – (59,78)
glycerol 50.0

Phosphite

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Phosphite is cheap and has been previously advocated as a suitable compound for the regeneration of nicotinamide cofactors in vitro. (28) Thermodynamically, the PO43–/HPO32– pair (E°′ = −0.65 V) is well below that of the NAD(P)+/NAD(P)H couple, resulting in an equilibrium constant (Keq ≈ 1011) strongly shifted toward the cofactor reduction at pH 7.0. (34) The redox reaction is catalyzed by phosphite dehydrogenase (PDH, EC 1.20.1.1), a homodimeric NAD+-dependent oxidoreductase (Figure 4A) well characterized from Pseudomonas stutzeri WM 88. (72) PDH is structurally related to the family of the D-2-hydroxyacid dehydrogenases, with which it shares the catalytic triad Arg-Glu-His in the active site for the hydride transfer (Figure 4B). (79) Although PDH shows a clear preference for NAD+ over NADP+ with 100 times higher catalytic efficiency (kcat/KM) for the coenzyme without the phosphate at the 2′ position, the cofactor specificity can be broadened through a double mutation (E175A/A176R), (80) allowing the reduction of both cofactors with a comparable kcat/KM, but slightly in favor of NADP+. The wild-type PDH has micromolar range affinity for both the native substrates, working efficiently in a pH range between 7.0 and 8.0 and below 40 °C (Table 2). The PDH activity is suppressed in the presence of sulfite in a similar concentration range (Ki = 16 μM) through a mechanism of competitive inhibition with the phosphite ion. The ionic strength of the reaction buffer is a crucial parameter to keep under control for optimal PDH activity, as suggested by the enzymatic inactivation observed by increasing the concentration of electrolytes such as sodium chloride. (72)

Figure 4

Figure 4. Phosphite dehydrogenase from P. stutzeri WM 88. (A) The homodimeric assembly of the holoenzyme in complex with NAD+ and sulfite, specific inhibitor of PDH (PDB entry: 4E5K). (B) A zoom into the PDH active site. The conserved catalytic triad Arg237, Glu266, and His292 allows catalysis with a proposed acid–base mechanism. The nucleophilic attack on the phosphite (here replaced by the competitive inhibitor sulfite) is initiated by histidine─via a water molecule─and is oriented by the combined action of arginine and glutamate, resulting in the reduction of the nicotinamide coenzyme.

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While there is in-depth biochemical and structural insight of the enzyme utilizing phosphite, knowledge of the membrane proteins that mediate its transport is still fragmentary. PtxABC and PhnCDE are the main transporters known to date that import phosphite. They have been identified from microorganisms capable of growing on the compound as the only source of phosphorus, as P. stutzeri WM 88, (81)Trichodesmium erythraeum IMS101, (82) and Prochlorococcus marinus. (83) Both PtxABC and PhnCDE are ATP-binding cassette (ABC) transporters composed of two nucleotide-binding subunits (PtxA or PhnC), two transmembrane subunits (PtxC or PhnE), and a solute-binding protein (PtxB or PhnD). The purified solute binding proteins have high affinities for phosphite with dissociation constant (KD) values in the nanomolar range. These proteins bind phosphate with much lower affinity (2–4 orders of magnitude higher KD depending on the specific protein). (84) Their crystallographic structures have elucidated the essential amino acids involved in the substrate recognition. In contrast, no functional information on either the ATP binding domains or the transmembrane domains of these ABC transporters is yet available in vitro, although the predicted structures of PtxA and PtxC from P. stutzeri are accessible in the AlphaFold database (AF-O69051-F1 and AF-O69053-F1, respectively). (85)
In view of the construction of a synthetic cell, the successful reconstitution of one among these phosphite transporters and the consequent use of PDH would not only provide reducing power by enabling the phosphite-consuming reaction, but it would also produce the phosphate ion. Phosphate is a ubiquitous osmolyte in living systems and an integral part of the main biomolecules (nucleic acids, proteins, lipids, carbohydrates). The design of a metabolic circuit that would couple the redox gain from phosphite oxidation with a phosphate sink (nucleosides, inositols, etc.) (86) would maximize the potential yield from both products of the PDH-mediated reaction, preventing the accumulation of an otherwise dead-end metabolite. However, likely the demand for phosphate is lower than its production. This would require export of phosphate for which different transporters are available. We will not elaborate here on phosphite/phosphate antiport─which would be a simple and elegant solution─as there is only a single membrane protein (PtdC from Desulfotignum phosphitoxidans) (87) which has been proposed as a phosphite/phosphate transporter, but no experimental evidence, neither in vivo nor in vitro, has been provided to date in support of this function.
There are a few alternatives based on known membrane transporters that can be implemented to remove the phosphate ion from the vesicular compartment (Figure 3A): first, the export of inorganic phosphate in combination with the removal of a proton (PitA from E. coli, (88) TCDB 2.A.20.1.1; PiPT from Piriformospora indica, (89) TCDB 2.A.1.9.10; PIC from pig mitochondria, (90) TCDB 2.A.29.4.2); second, the symport of phosphate and sodium ion out of the lumen (YjbB from E. coli, (91) TCDB 2.A.58.2.1); third, the facilitated diffusion of inorganic phosphate alone (Pho1 from Arabidopsis thaliana, (92) TCDB 2.A.94.1.1). With the exception of the purified and biochemically active PIC (90,93) and the crystallized PiPT, (89) all the other mentioned candidate transporters have not been characterized in vitro, but only partially in vivo. Considering the nontrivial troubleshooting involving the overproduction, purification and functional characterization (lipid and detergent requirements, membrane orientation, activity assays, etc.) (94−96) of membrane proteins, the choice of the phosphite/phosphate pair could require substantial optimization work. Thus, the biochemical and structural gap constitutes currently one of the major limitations to be addressed in order to apply phosphite and phosphate transporters in bottom-up systems such as artificial cells. In the absence of a functionally characterized phosphite/phosphate antiporter, this redox couple would depend on ATP for the phosphite import via the ABC-transporters; typically a synthetic cell would be equipped with an ATP-regeneration module, but this redox system would increase the demand from that module.

Formate

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While formate in high concentrations can be toxic to some living organisms, (97,98) it represents a harmless and highly soluble C1 compound to readily provide reducing power to artificial cells designed in a bottom-up fashion. Since it does not require a membrane transporter to cross phospholipid bilayers in the millisecond time scale, (99) formate provides the advantage of requiring exclusively a NAD(P)+-dependent formate dehydrogenase (FDH) to reduce the nicotinamide cofactor, while CO2 is formed as a reaction product. NAD(P)+-dependent FDHs (EC 1.17.1.9) can be subdivided in metal-containing FDHs and nonmetal FDHs. (100) Metal-containing FDHs are heteromeric complexes displaying a huge diversity in the overall structure (number of subunits, amount of embedded iron–sulfur clusters, presence of flavin mononucleotide as prosthetic group), but a high degree of conservation in correspondence of the active site embedding molybdenum (Mo) or tungsten (W). (100,101) Regardless of the metal nature, Mo/W is always coordinated by two molybdopterin cofactors, a (seleno)cysteine and a terminal sulfo-group. This abundance of cofactors to be kept properly embedded and in the appropriate redox state for optimal protein activity makes metal-containing FDHs challenging for straightforward purification and storage; therefore, we consider them not very suitable for synthetic bottom-up systems.
On the other side, nonmetal FDHs are well characterized and easy to produce recombinantly to high purity and yield. The characterization of more than a dozen different homologues (bacterial and yeast) offers a wide selection of FDHs to draw upon to suit particular kinetic or functional requirements (e.g., chemical and thermal stability). (74) Nonmetal FDHs are homodimers (Figure 5A) around 90 kDa with wide pH optimum, high stability above 50 °C, affinity for formate in the low millimolar range and kcat values that do not exceed 10–15 s–1. Although strictly dependent on NAD+, their cofactor specificity can be switched with NADP+ with a single mutation of aspartate 221 to either alanine, glycine, serine, or glutamine (D221A/G/S/Q), (104) as the negative charge of the aspartate which repels the phosphate group of NADP+ is removed. Several studies have shown that up to five additional mutations can improve the catalytic efficiency for NADP+, while NAD+ further becomes a poorer cosubstrate. (104−106)

Figure 5

Figure 5. Formate dehydrogenase from Pseudomonas sp. 101. (A) The nonmetal containing FDH is homodimeric, here shown in complex with NAD+ and azide (N3), an inhibitor analogue of formate (PDB entry: 2NAD). (B) The catalysis mediated by FDH requires the involvement of multiple amino acids. His332 and Ser334 establish hydrogen bonds with the carboxamide group of the nicotinamide ring. Through a hydrogen bond with His332, Gln313 plays a key role in the FDH reaction, ensuring its characteristic wide pH optimum. (102) The binding with formate (here replaced with the analogue, azide) is established through electrostatic interactions with the positively charged substituents of Arg284 and Asn146, while Ile122 favors the charge counterbalance of the substrate. The hydrophobic cluster (Phe97 and Pro98) promotes the spatial constriction and consequent destabilization of formate into the configuration of the reaction intermediate. Distances and other amino acids involved to a minor extent in the catalytic mechanism are here omitted, but discussed in detail somewhere else. (73,103)

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The proposed mechanism for formate oxidation involves first the correct orientation and trapping of the nicotinamide cofactor via the concerted action of several amino acids (Figure 5B), then attacking the formate molecule through electrostatic interactions with His332 and Arg284, and then concluding the catalytic cycle by releasing the reaction products NADH and CO2, restoring the original conformation of the active site (thoroughly discussed in the thematic reviews (73,100)).
The use of a weak acid such as formic acid to fuel synthetic cells with reducing power would lead to luminal acidification, because it is the undissociated form (formic acid) that crosses the membrane, and then dissociates back into formate plus a proton. (99) Nonetheless, a sufficient buffer capacity could attenuate such change in internal pH.
The enzymatic oxidation of formate fully meets the requirements for integration into a minimal compartmentalized metabolism, as the reaction product (CO2) diffuses out of the liposomes and only the reducing equivalents are retained within the lumen(s) in the form of NAD(P)H. We recently demonstrated the applicability of formate as a suitable electron donor using it to feed synthetic cells (both large and giant unilamellar vesicles) with reducing power via NAD(P)+ cofactors. (14) The liposomal reconstitution of a nonmetal formate dehydrogenase enabled the activity of a minimal enzymatic pathway transferring hydrides to an electron sink of biological relevance for its antioxidant function, namely glutathione.

Molecular Hydrogen

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Unlike the other electron donor candidates described so far, H2 is highly insoluble. Although an estimate of the P value for dihydrogen has not yet been reported for phospholipid membranes, it is reasonable to assume it is in the range as for other small gaseous molecules such as O2, N2, CO, CO2, NO2 (1–5 cm/s). (107) The enzymatic oxidation of H2 into protons and electrons can be performed by three different types of hydrogenases ([NiFe], [FeFe], and [Fe]), (108) but only those that incorporate the nickel ion (EC 1.12.1.2) are soluble proteins able to couple the dihydrogen breakdown with the reduction of NAD(P)+ cofactors. Compared to hydrogenases using exclusively iron as a metal ion cofactor, soluble [NiFe] hydrogenases also show greater tolerance to oxygen, (109) a property that makes them highly versatile enzymes for biocatalysis, as well as synthetic biology.
Since soluble [NiFe]-hydrogenases (SHs) encompass a diverse group of heteromeric metalloproteins, we will here describe only the most studied homologue from Cupriavidus necator H16 (formerly known as Ralstonia eutropha) (110) as a representative model for SHs. As illustrated in Figure 6A, they have a tetrameric active core composed of the hydrogenase (HoxHY) and the NAD(P)+-diaphorase (HoxFU) modules, along with two additional identical subunits (HoxI2), introducing a further nucleotide-binding site to the protein complex. (76) The coordination of the metals of the [NiFe] active center, in the HoxH subunit, is mediated by cysteine residues for the nickel ion and by the nonprotein ligands CO and cyanides (CN–) for the iron ion. The electron transfer from H2 to NAD+ is made possible by the further incorporation of 2 molecules of flavin mononucleotide (FMN-a and FMN-b), respectively in HoxY and HoxF, and several iron–sulfur clusters along the tetrameric complex (one [4Fe-4S] in HoxY, two [4Fe-4S] and one [2Fe-2S] in HoxU, one [4Fe-4S] in HoxF). The 2.6 Å tetrameric structure (Figure 6B) of the SH from Hydrogenophilus thermoluteolus TH-1 (approximately 40% sequence identity with the SH of C. necator) currently offers the most complete model for a mechanistic understanding of the SH family. (111)

Figure 6

Figure 6. Soluble [NiFe]-hydrogenase structure. (A) The scheme of the subunit and cofactor assembly. The hydrogenase module HoxHY initiates the dihydrogen oxidation (HoxH in blue, HoxY in cyan). Different iron–sulfur clusters (red and yellow balls) allow the electron transfer from the hydrogenase to the diaphorase subunit HoxFU (HoxU in gray, HoxF in green), leading to the NAD+ reduction into NADH. The binding of 2 identical HoxI subunits (in yellow) to HoxF generates an hexameric assembly that promotes the reductive-protein activation by NADPH. (B) The tetrameric structure of SH from Hydrogenophilus thermoluteolus TH-1 (PDB entry: 5XF9). The color scheme is the same used in A.

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In terms of applicability, the kinetic parameters of SHs are favorable, as they show high affinity for both the substrates, as well as a turnover number greater than 100 molecules converted in one second. The pH optimum of SH corresponds to 8.0 and could be maintained in both potassium phosphate (KPi) and Tris buffers, although only the tetrameric form is stable in the latter buffer. The ionic strength of the reaction medium is also a crucial parameter for enzymatic functionality, with the protein being inhibited by more than 70% above 200 mM KPi. (75) Importantly, unless anaerobic conditions are ensured thorough the whole isolation procedure, the purified SH requires a short reducing pretreatment (with more than 5 μM NADH) to be active in both the conformations of tetramer or hexamer, as the enzyme is inactivated over time when in contact with the air dioxygen. (76) A recent insight on the active site of SH of H. thermoluteolus suggested a protective role of Glu32 from oxidative damage, (112) preventing the access of dioxygen to the embedded and cysteine-coordinated nickel ion; further exploration on the mechanism of oxygen tolerance is needed to promote a broader biotechnological use of SHs. In-depth studies on the cofactor specificity have not been conducted so far on SHs, but a first insight was provided by the work of Preissler and colleagues: (113) they relaxed the strict NAD+-dependence of SH isolated from C. necator via a double mutation E341A/S342R that allowed to recognize also NADP+ as a substrate with similar affinity to NAD+, although with a four times lower turnover number.
Similarly to formic acid, also the use of the dihydrogen oxidation to feed reductive reactions would not affect the carbon balance of the metabolism, as only one proton is released into the compartment while NAD(P)H is formed. But unlike a weak acid, H2 permeation does not lead to luminal acidification or water efflux, maintaining the original pH and volume at which our synthetic cells would operate their metabolic functions. The advantages that reaction products do not have to be disposed of and dihydrogen is readily membrane permeable are somewhat counterbalanced by the multiplicity of subunits and prosthetic groups required for the functionality of SHs: the protocols for the purification and activity of these dehydrogenases are less trivial than the other dehydrogenases here described, but in any case feasible with the adequate procedures extensively reported elsewhere. (108,114,115)

Glycerol

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Glycerol is a polar and nontoxic triol able to diffuse across phospholipid membranes (P = 2 × 10–5 cm/s in DOPC). (48) These characteristics make it a suitable substrate to be exploited for the bottom-up assembly of synthetic cells, as already shown for the synthesis of the phospholipid precursor glycerol-3-phosphate within liposomes fed in a continuous-flow dialysis apparatus. (12) In addition to the supply of carbon for assimilation, glycerol can also provide reducing equivalents to NAD(P)+ cofactors. The NAD+-dependent glycerol dehydrogenase (GlyDH, EC 1.1.1.6) catalyzes the oxidation of glycerol into dihydroxyacetone, and the enzyme can also oxidize glycerol-derivatives albeit at a significantly lower rate. (78) The recognition of a broad range of substrates is a well-known characteristic of the heterogeneous group of polyol dehydrogenases: the same applies to GlyDH, considered as the representative model of the family III of polyol dehydrogenases. (59) Biochemical understanding on GlyDHs is based on the studies carried out on the protein isolated from Geobacillus stearothermophilus. (116) Its kinetic parameters are strongly affected by the pH of the reaction medium, revealing better KM and kcat values at alkaline pH. Inactivation by chelating agents provided the first evidence on a strict dependency of GlyDH on zinc (Zn2+), later proved to be embedded in the active site of the protein. In fact, crystallographic structures in the apo- and holo-forms (59) contributed to shed light not only on the oligomeric state (Figure 7A), but also on the mechanistic basis behind the metal coordination and the catalytic action of the enzyme (Figure 7B). Three specific amino acids (Asp173, His256, and His274) and a water molecule allow the tetrahedral coordination of Zn2+ to the protein active site. Other key amino acids correctly orient the substrates forming hydrogen bonds with the nicotinamide ring and van der Waals interactions with glycerol. Studies on the cofactor specificity for GlyDH are not available but, looking at its holo-structure in the presence of NAD+ (similarly to what observed in nonmetal FDHs with Asp221), an aspartate in position 39 lies in the proximity of the 2′-OH group of the adenine ribose forming a hydrogen bond with the coenzyme: the mutagenesis of Asp39 (and neighboring amino acids) could therefore be investigated to develop a NADP+-dependent GlyDH.

Figure 7

Figure 7. Glycerol dehydrogenase from Geobacillus stearothermophilus. (A) The enzyme has an homo-octameric assembly in solution, here illustrated in complex with NAD+ (PDB entry: 1JQ5). (B) Overview of the active site of GlyDH in complex with the substrates. By superimposing the structures in complex with NAD+ and glycerol (PDB entries: 1JQ5, 1JQA), the essential amino acids to accommodate both substrates are highlighted. Asp100, Ser127, and Leu129 establish a hydrogen bond network with the carboxamide group of the nicotinamide moiety. Glycerol is correctly oriented toward the catalytic center through van der Waals interactions between Phe247 and the carbon atoms of the substrate. The coordination of zinc is mediated by dipole interactions with Asp173, His256, His274, and a water molecule.

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The choice of glycerol to feed synthetic cells with reducing equivalents can be beneficial on multiple levels. In addition to bypassing the need for membrane proteins, it can increase the operational stability of the compartmentalized metabolism: glycerol is a stabilizing agent for numerous proteins, allowing to prolong the enzymatic functionality over time. (117) While the glycerol that does not react stoichiometrically with the cofactors can boost metabolic stability, the reaction product dihydroxyacetone can potentially permeate across phospholipid membranes without the support of a protein translocator. Recent studies on bacterial and archaeal lipid membranes have in fact highlighted membrane permeability comparable to both glycerol and dihydroxyacetone, (118) suggesting not too significant differences between their permeability rates. The luminal entry of glycerol, followed by the diffusion out of its oxidation product, maintains unaltered the carbon mass of the reactants enclosed in the vesicles, resulting in the orthogonal provision of reducing equivalents to downstream NAD(P)H-dependent reactions.

Enzymatic Transhydrogenation: A Carbon-Free Approach to Tackle the Cofactor Specificity Bottleneck

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While NAD(H) is mostly linked to energy provision in combination with ATP generation during the catabolic breakdown of high-energy compounds, (119) NAD(P)H plays a main role in biosynthetic and antioxidant pathways. (120) The overview of suitable dehydrogenases presented above showcases how cofactor specificity represents a key feature of redox enzymes. Multiple structural studies combined with protein engineering efforts have been contributing to overcome this limitation by changing or loosening the cofactor preference of specific oxidoreductases, (80,121−123) but some dehydrogenases (SHs, GlyDHs) are still not extensively characterized with respect to cofactor specificity. More importantly, even when it is possible to reduce both coenzymes through the action of native or mutant (PDHs, FDHs, SHs) enzymes, it is likely that the demand for two reduced cofactors will not be constant over the lifetime of a synthetic cell, thus the ability of transferring reducing equivalents between the two compounds is desirable. Specific pyridine nucleotide transhydrogenases (THs) would allow to transfer the reducing equivalents from one reduced cofactor to another oxidized one, cycling the redox status of the nicotinamides and therefore to link the different pathways of the reductive metabolism dependent on both NADH and NADPH (Figure 8A). THs exist as membrane (mTHs) or soluble (sTHs) enzymes catalyzing the reversible hydride transfer among cofactors, although with very different mechanisms. (124) Their integration in a minimal metabolism may provide a useful alternative to overcome the barrier imposed by cofactor specificity, while preventing the requirement for extra metabolic modules to regenerate the redox status of both nicotinamide cofactors via formation of new metabolites (mostly carbon-based) that should then be internally recycled or exported outside of the vesicular lumen. We now proceed to briefly describe the main biochemical and structural features of both types of THs characterized to date.

Figure 8

Figure 8. Transhydrogenases in synthetic cells. (A) Scheme of the redox regeneration. Following the luminal access of the electron donor (passively indicated by the dashed arrow, actively by the solid arrow), NAD+ (left side) or NADP+ (right side) is reduced by an upstream specific dehydrogenase. A downstream transhydrogenase transfers electrons from one cofactor to another, overcoming the limitation of cofactor specificity, and reoxidizing the cosubstrate NAD(P)H during transhydrogenation. An additional sink enzymatic reaction can take advantage of the new reduced cofactor, while pulling the transhydrogenase reaction out of equilibrium. (B) Structural organization of membrane transhydrogenases. On top, the scheme of the protein assembly. All known mTHs are organized into three distinct domains: dI, NAD(H)-binding in yellow; dII, transmembrane in cyan; dIII, NADP(H)-binding in red. On bottom, the heteromeric structure of the holo-enzyme NNT from Ovis aries mitochondria (PDB entry: 6QTI) in complex with NAD+ and NADP+. (C) Structural and catalytic insight of soluble transhydrogenases. On the left, the monomeric predicted structure of the soluble transhydrogenase from E. coli K-12 (AlphaFold entry: AF-P27306) embedding the prosthetic group─FAD─upon superimposition with the related lipoamide dehydrogenase from Thermus thermophilus HB8 (PDB entry: 2EQ7). On the right is a zoom into the FAD-binding domain in complex with NAD+, highlighting cysteine 45 and threonine 50, as they are conserved amino acids in sTHs for efficient catalytic activity.

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Membrane Transhydrogenases

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The membrane transhydrogenase family (TCDB 3.D.2, also known as proton-translocating transhydrogenase family) includes proteins partially embedded in the phospholipid bilayer that protrude a catalytically active domain into the cytosol (in bacteria) or the mitochondrial matrix (in eukarya) to catalyze transhydrogenation. The hydride transfer from NAD(P)H to NAD(P)+ is coupled to the translocation of a proton across the membrane (Figure 8B). In vivo, the normal ratios of NADH/NAD+ and NADP+/NADPH favor the transhydrogenation toward the formation of NADPH, which is then released from the dIII domain into the cytosol by importing a proton (available from the physiological electrochemical gradient). (125) Nonetheless, the reverse reaction (NADH-forming) can also be mediated by mTHs exporting a proton, as demonstrated in vitro in submitochondrial particles. (126) The overall structural organization of mTHs is strongly conserved, although they can consist of 1 to 3 polypeptide chains (e.g., 1 in mammalian mitochondria, 2 in E. coli, 3 in Thermus thermophilus). (127) The mTHs are natively homodimers; each protomer can be divided into 3 distinct domains (Figure 8B): the most membrane-distal dI (specific for NAD(H)-binding), the central transmembrane site dII, and the globular domain most proximal to the membrane dIII (binding NADP(H)). Structural studies on the mammalian and T. thermophilus mTHs proposed a common “antiphase” catalytic mechanism (also described as “division of labor”) (128,129) in which the two dIII protomers alternate opposite “face-up” and “face-down” conformations: the “face-up” conformation is needed for hydride transfer, while the “face-down” conformation allows proton translocation across the membrane.
Biochemically, the best characterized mTHs are NNT from Bos taurus mitochondria and PntAB from E. coli. (130,131) Upon purification, they both have similar high affinities for oxidized and reduced nicotinamide cofactors (KM values between 2 and 150 μM) and comparable turnover numbers around 10–15 s–1. The mTH-mediated transhydrogenation seems to be easily integrated with further essential biological processes in vitro: the recent work by Graf et al. (131) demonstrated how purified PntAB can work synergistically with ATP synthase, driving the synthesis of ATP through the generation of a proton motive force at the expense of the transhydrogenation from NADPH to NAD+. Nonetheless, the activity of PntAB in proteoliposomes mostly takes place─and is thus measured─extraluminally as it assumes a preferential orientation (∼75%) with the NAD(P(H))-binding domains extruding from the outer surface of the vesicles. Therefore, methods that promote correct orientation to ensure transhydrogenation in the vesicular lumen are desirable for efficient utilization of mTHs in synthetic cells. Alternatively, strategies to remove traces of external protein activity could be applied (e.g., enzymatic scavenger systems (14)).

Soluble Transhydrogenases

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The sTHs (EC 1.6.1.1) are energy-independent flavoproteins that tightly bind FAD as prosthetic group to perform transhydrogenation between nicotinamide cofactors. (124) Although they are mainly associated with the maintenance of the NADPH/NADP+ pool by reoxidizing the surplus of cytosolic NADPH, (132) sTHs can reversibly mediate transhydrogenation both in vivo and in vitro.
The most studied sTHs belong to Pseudomonas fluorescens, Azotobacter vinelandii and E. coli (each of them annotated as SthA), with the first 2 orthologs displaying high sequence identity (85%) with each other; SthA from E. coli is instead more evolutionarily distant from the other 2 sTHs, with a degree of identity that does not exceed 60% and 64%, respectively. Interestingly, despite the homology the proteins have very different oligomeric conformations. Electron microscopy studies have shown filaments up to 300 nm in length for SthA from A. vinelandii, and nearly one micrometer long for SthA from P. fluorescens, the functional unit of which is assumed to be an octamer. (133,134) In E. coli, SthA adopts a globular conformation characterized by 8 protomers with an overall radius around 8 nm. (135) High resolution structures of the octameric complex have not yet been resolved for any sTH homologue, but the predicted monomer structure (Figure 8C, left panel) is available on the AlphaFold database.
Assuming the size of metabolically active synthetic cells in a range between 1 and 10 μm in diameter, (42) we consider the homo-octameric SthA from E. coli more suitable than the filamentous ones to guarantee the shuttle of hydrides between cofactors in a compartmentalized metabolism; in this way it would be possible to minimize the effects of excluded volume, and consequently preserve useful luminal space to encapsulate further metabolic modules. (51) We therefore focus on the biochemical features of SthA from E. coli as a representative model for the biochemistry of sTHs.
The purified SthA catalyzes the concomitant oxidation of NADPH and reduction of the non-natural substrate thio-NAD+ with a turnover number between 170 and 260 s–1 and KM values around one hundred micromolar for both substrates. (136) The SthA-mediated reaction consuming NADH while reducing thioNADP+ has also been recently characterized, revealing a lower kcat value of 10–15 s–1, and higher affinity for thioNADP+ compared to NADH (0.1 and 2.6 mM, respectively). (135) Substrate inhibition occurs in both directions of reaction with NADPH, NADH and thioNADP+, as well as high concentrations of phosphate ion decrease the activity of SthA to 15–25% compared to a phosphate-free reaction medium. The enzyme works optimally at pH 7.5–8.5 and at a temperature of 35 °C, maintaining full activity for at least 3 weeks when incubated at 4 °C.
sTHs are related to the disulfide reductase family of flavoproteins, and retain an exceptional CXXXXT domain (instead of the typical CXXXXC domain for reducing disulfides) which promotes tight FAD-embedding to the active site (Figure 8C, right panel) and allows for efficient transhydrogenation. (135) As reported for several flavoproteins, SthA also exhibits oxidase activity both in the absence of an oxidized cofactor as acceptor and during transhydrogenation. This reactivity with dioxygen leads to a marginal formation of hydrogen peroxide and superoxide anion (together they intercept 2% of reducing equivalents from NADH to thioNADP+, while the remaining 98% is used for transhydrogenation). Although small, the formation of such side products should be taken into account in the design of a “in confinement” enzymatic network, as they could react and eventually damage membranes, (deoxy)ribonucleic acids, and other enzymes. (137) The addition of antioxidant systems such as glutathione reductase/peroxidase, catalase, or superoxide dismutase would overcome the problem by forming harmless molecules such as reduced glutathione or water.

Conclusions

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The pivotal role of NAD(H) and NADP(H) for redox metabolism requires strategies to reduce the cofactors by external electron donors within artificial cells to efficiently feed any other reductive reaction in a controllable manner. By establishing parameters for the reduction of nicotinamides enclosed by selectively permeable vesicles, we proposed four possible candidates as suitable electron donors. Our analysis employed an “enzymatic” view (dehydrogenases and membrane transporters) by reviewing the structural and biochemical properties of the protein biocatalysts transporting or oxidizing these compounds for the downstream reduction of NAD(P)+ cofactors. Considering proteins as the crucial catalysts for the functionality of metabolic networks, keeping a system with life-like properties out of thermodynamic equilibrium, we advocate for an in-depth understanding of the chosen biocatalyst(s) for robust activity in minimal synthetic cells.
From our work it emerges that each of the considered redox donors exhibits attractive properties in some respects, but certain limitations in others. Phosphite demands a membrane transporter for uptake (and the product phosphate requires a dedicated export system) and it suffers from a lack of purified proteins with available characterization. Membrane-permeable donors show different side effects: luminal acidification in the case of formate permeation, relative oxygen sensitivity for the enzyme(s) utilizing molecular hydrogen, relatively slow diffusion in and out the compartment for glycerol and its oxidized product (dihydroxyacetone). For enzymes in particular, it is also important to consider whether they need to incorporate one or more prosthetic groups to perform their function. An enzyme embedding multiple metal ions (e.g., [NiFe]-hydrogenase) is less trivial to work with than metal-free proteins, and much less versatile during the steps of protein purification, vesicular encapsulation, and in vitro activity. More importantly, the cofactor specificity of dehydrogenases appears as a recurring element that changes and at the same time distinguishes each specific homologue. Transhydrogenases are solutions to overcome this issue through the hydride transfer between NAD(H) and NADP(H), with the benefit of avoiding the direct involvement of other carbon-based metabolites.
Likely many efforts focused on constructing synthetic cells will include the establishment of NADPH-dependent biosynthetic pathways, requiring a sufficiently high ratio of NADPH/NADP+ to push their reactions. As many available donor systems primarily generate NADH, the use of transhydrogenases is a promising way of conveying the reducing power from one cofactor to another. The membrane-bound transhydrogenases are prime candidates to convert NADH into NADPH at the expense of proton translocation across the membrane. The energy investment will help to overcome the thermodynamic gap of transferring a hydride from NADH, upward to maintaining a high NADPH/NADP+ ratio to push biosynthesis. This proton motive force can be provided for example by ATP synthase or proton-pumping rhodopsin, as already demonstrated in liposome systems. (13)
For thermodynamically very favorable electron donating substrates (e.g., phosphite, formate, and hydrogen), the NADH/NAD+ ratio that can be generated by the electron donor may be much higher than the typical ratio found in most bacteria (0.03–0.32). (22) In this case, a non-energy-consuming soluble transhydrogenase may be sufficient to drive the hydride transfer from NADH to NADP+: SthA from E. coli has been employed recently for this purpose in synthetic cells in synergy with formate as electron donating substrate and glutathione as electron sink. (14)
To conclude, the selection of an electron donor should be made on the basis of the experimental conditions of the metabolic processes to be integrated in cell-like systems, tailoring the choice of the most suitable protein homologue so that it can function in optimal conditions, without interfering with other encapsulated components. In this regard, protein engineering is a fruitful resource available to the scientific community to broaden the range of scenarios in which a protein can be used (different optimum of pH and temperature, stability in selected buffers, better kinetics, substrate specificity). With further studies to elucidate the structure, and therefore the biochemistry of more and more enzymes, we will be able to customize their utilization in support of bottom-up synthetic biology.
Our recently demonstrated redox regeneration system based on formic acid may serve as a blueprint to develop redox homeostasis for synthetic cells. This system, or alternative systems, following the guidelines provided in this work, can support the further construction of more complex metabolisms in bottom-up synthetic cells.

Author Information

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  • Corresponding Author
  • Authors
    • Michele Partipilo - Department of Biochemistry, Groningen Institute of Biomolecular Sciences & Biotechnology, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands;  Orcidhttps://orcid.org/0000-0003-4150-2341
    • Nico J. Claassens - Laboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE Wageningen, The Netherlands;  Orcidhttps://orcid.org/0000-0003-1593-0377
  • Author Contributions

    M.P. wrote the draft manuscript. N.J.C. and D.J.S. provided extensive feedback. All authors contributed and approved the final version of the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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The authors thank Michiel Punter for the acquisition of the complete dataset of metabolites and respective reactions available on the public online database Escherichia coli Metabolome Database. The main author is grateful to Mr. Douglas Adams for the series of books that inspired the title and somehow triggered the initial idea behind this manuscript. This work was supported by the Dutch Research Council (NWO) Gravitation program (Building a Synthetic Cell), Grant No. 024.003.019.

Abbreviations

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NAD+

oxidized nicotinamide adenine dinucleotide
NADH

reduced nicotinamide adenine dinucleotide
NADP+

oxidized nicotinamide adenine dinucleotide phosphate
NADPH

reduced nicotinamide adenine dinucleotide phosphate
ATP

adenosine triphosphate
E°′

standard potential reduction
CO

carbon monoxide
CO2

carbon dioxide
H2

molecular hydrogen
H+

hydrogen ion
FAD

oxidized flavin adenine dinucleotide
HCOO–

formate or anionic form of formic acid
DOPC

1,2-dioleoyl-sn-glycero-3-phosphocholine
EC

enzyme commission
GDH

d-glucose dehydrogenase
SO42–

sulfate
HSO32–

sulfite
CODH

carbon monoxide dehydrogenase
ABC-transporter

ATP-binding cassette transporter
ADP

adenosine diphosphate
Na+

sodium ion
TX-TL

transcription–translation
NiFe

nickel–iron
P

permeability coefficient
PO43–

phosphate
HPO32–

phosphite
Keq

equilibrium constant
PDH

phosphite dehydrogenase
kcat

turnover number
KM

Michaelis–Menten constant
PDB

Protein Data Bank
KD

dissociation constant
TCDB

transporter classification database
FDH

formate dehydrogenase
Mo

molybdenum
W

tungsten
N3

azide
Fe

iron
O2

dioxygen
N2

nitrogen gas
NO2

nitrite
SH

soluble [NiFe]-hydrogenase
CN–

cyanide
FMN

flavin mononucleotide
KPi

potassium phosphate
Tris

tris(hydroxymethyl)aminomethane
GlyDH

glycerol dehydrogenase
Zn2+

zinc ion
TH

pyridine nucleotide transhydrogenase
mTH

membrane transhydrogenase
sTH

soluble transhydrogenase.

References

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This article references 137 other publications.

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    Micelles that can catalyze their replication have been described recently. In the previous expts., micelles (or bilayer vesicles) were always present in the initial reaction mixt., i.e., the system was presented with the bounded structures required for autocatalysis. Here a system is described in which autocatalytic micelles are formed from amphiphiles that are themselves generated from a hydrolysis reaction in the absence of compartmental structures. Alk. hydrolysis of Et caprylate (itself insol. in water) yields sodium caprylate, initially at a very slow rate; but as soon as sufficient caprylate is formed for aggregation into micelles to take place, there is an exponential increase in reaction rate owing to micellar catalysis. These self-assembling surfactant structures may consequently provide a model system for studies of prebiotic chem. The possible relevance of this process to prebiotic chem. is emphasized by the observation that the micelles can be converted into more-robust vesicles by a pH change induced by dissolved CO2.
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  2. 2
    Walde, P.; Wick, R.; Fresta, M.; Mangone, A.; Luisi, P. L. Autopoietic Self-Reproduction of Fatty Acid Vesicles. J. Am. Chem. Soc. 1994, 116 (26), 11649– 11654,  DOI: 10.1021/ja00105a004
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    2
    Autopoietic Self-Reproduction of Fatty Acid Vesicles
    Walde, Peter; Wick, Roger; Fresta, Massimo; Mangone, Annarosa; Luisi, Pier Luigi
    Journal of the American Chemical Society (1994), 116 (26), 11649-54CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Conditions are described under which vesicles formed by caprylic acid and oleic acid in H2O are able to undergo autopoietic self-reprodn.-e.g. an increase of their population no. due to a reaction which takes place within the spherical boundary of the vesicles themselves. This is achieved by letting a certain amt. of the neat H2O-insol. caprylic or oleic anhydride hydrolyze at alk. pH: the initial increase of the concn. of the released acid/carboxylate is extremely slow (several days to reach the conditions for spontaneous vesicle formation), but afterwards, the presence of vesicles brings about a rapid 2nd phase leading to more and more vesicles being formed in an overall autocatalytic process. The catalytic power of the caprylic acid and oleic acid vesicles toward the hydrolysis of the corresponding anhydride is documented in a set of independent expts. In these expts., the hydrolysis was carried out in the presence of vesicles at a pH corresponding approx. to the pK of the acid in the vesicles. The process of autopoietic self-reprodn. of caprylic acid and oleic acid vesicles is studied as a function of temp.: by increasing temp. (≤ 70°), the exponential time progress of vesicle formation tends to become steeper while the long initial slow phase is significantly shortened. The caprylic acid and oleic acid vesicles are characterized by electron microscopy and by detg. their internal vol. The question whether and to what extent these vesicles form a classic chem. equil. system (e.g., in which the free surfactant is in equil. with the aggregates) is also studied.
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  3. 3
    Noireaux, V.; Libchaber, A. A Vesicle Bioreactor as a Step toward an Artificial Cell Assembly. Proc. Natl. Acad. Sci. U. S. A. 2004, 101 (51), 17669– 17674,  DOI: 10.1073/pnas.0408236101
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    3
    A vesicle bioreactor as a step toward an artificial cell assembly
    Noireaux, Vincent; Libchaber, Albert
    Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (51), 17669-17674CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    An Escherichia coli cell-free expression system is encapsulated in a phospholipid vesicle to build a cell-like bioreactor. Large unilamellar vesicles contg. exts. are produced in an oil-ext. emulsion. To form a bilayer the vesicles are transferred into a feeding soln. that contains ribonucleotides and amino acids. Transcription-translation of plasmid genes is isolated in the vesicles. Whereas in bulk soln. expression of enhanced GFP (eGFP) stops after 2 h, inside the vesicle permeability of the membrane to the feeding soln. prolongs the expression for up to 5 h. To solve the energy and material limitations and increase the capacity of the reactor, the α-hemolysin pore protein from Staphylococcus aureus is expressed inside the vesicle to create a selective permeability for nutrients. The reactor can then sustain expression for up to 4 days with a protein prodn. of 30 μM after 4 days. Oxygen diffusion and osmotic pressure are crit. parameters to maintain expression and avoid vesicle burst.
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  4. 4
    Kita, H.; Matsuura, T.; Sunami, T.; Hosoda, K.; Ichihashi, N.; Tsukada, K.; Urabe, I.; Yomo, T. Replication of Genetic Information with Self-Encoded Replicase in Liposomes. ChemBioChem. 2008, 9 (15), 2403– 2410,  DOI: 10.1002/cbic.200800360
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    Replication of genetic information with self-encoded replicase in liposomes
    Kita, Hiroshi; Matsuura, Tomoaki; Sunami, Takeshi; Hosoda, Kazufumi; Ichihashi, Norikazu; Tsukada, Koji; Urabe, Itaru; Yomo, Tetsuya
    ChemBioChem (2008), 9 (15), 2403-2410CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    In all living systems, the genome is replicated by proteins that are encoded within the genome itself. This universal reaction is essential to allow the system to evolve. Here, we have constructed a simplified system involving encapsulated macromols. termed a "self-encoding system", in which the genetic information is replicated by self-encoded replicase in liposomes. That is, the universal reaction was reconstituted within a microcompartment bound by a lipid bilayer. The system was assembled by using one template RNA sequence as the information mol. and on in vitro translation system reconstituted from purified translation factors as the machinery for decoding the information. In this system, the catalytic subunit of Qβ replicase is synthesized from the template RNA that encodes the protein. The replicase then replicates the template RNA that was used for its prodn. This in-liposome self-encoding system is one of the simplest such systems available; it consists of only 144 gene products, while the information and the function for its replication are encoded on different mols. and are compartmentalized into the microenvironment for evolvability.
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  5. 5
    Luisi, P. L.; Ferri, F.; Stano, P. Approaches to Semi-Synthetic Minimal Cells: A Review. Naturwissenschaften 2006, 93 (1), 1– 13,  DOI: 10.1007/s00114-005-0056-z
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    Approaches to semi-synthetic minimal cells: a review
    Luisi, Pier Luigi; Ferri, Francesca; Stano, Pasquale
    Naturwissenschaften (2006), 93 (1), 1-13CODEN: NATWAY; ISSN:0028-1042. (Springer)
    Following is a synthetic review on the minimal living cell, defined as an artificial or a semi-artificial cell having the minimal and sufficient no. of components to be considered alive. We describe concepts and expts. based on these constructions, and we point out that an operational definition of minimal cell does not define a single species, but rather a broad family of interrelated cell-like structures. The relevance of these researches, considering that the minimal cell should also correspond to the early simple cell in the origin of life and early evolution, is also explained. In addn., we present detailed data in relation to minimal genome, with observations cited by several authors who agree on setting the theor. full-fledged minimal genome to a figure between 200 and 300 genes. However, further theor. assumptions may significantly reduce this no. (i.e. by eliminating ribosomal proteins and by limiting DNA and RNA polymerases to only a few, less specific mol. species). Generally, the exptl. approach to minimal cells consists in utilizing liposomes as cell models and in filling them with genes/enzymes corresponding to minimal cellular functions. To date, a few research groups have successfully induced the expression of single proteins, such as the green fluorescence protein, inside liposomes. Here, different approaches are described and compared. Present constructs are still rather far from the minimal cell, and exptl. as well as theor. difficulties opposing further redn. of complexity are discussed. While most of these minimal cell constructions may represent relatively poor imitations of a modern full-fledged cell, further studies will begin precisely from these constructs. In conclusion, we give a brief outline of the next possible steps on the road map to the minimal cell.
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    Forster, A. C.; Church, G. M. Towards Synthesis of a Minimal Cell. Mol. Syst. Biol. 2006, 2 (1), 45,  DOI: 10.1038/msb4100090
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    6
    Towards synthesis of a minimal cell
    Forster Anthony C; Church George M
    Molecular systems biology (2006), 2 (), 45 ISSN:.
    Construction of a chemical system capable of replication and evolution, fed only by small molecule nutrients, is now conceivable. This could be achieved by stepwise integration of decades of work on the reconstitution of DNA, RNA and protein syntheses from pure components. Such a minimal cell project would initially define the components sufficient for each subsystem, allow detailed kinetic analyses and lead to improved in vitro methods for synthesis of biopolymers, therapeutics and biosensors. Completion would yield a functionally and structurally understood self-replicating biosystem. Safety concerns for synthetic life will be alleviated by extreme dependence on elaborate laboratory reagents and conditions for viability. Our proposed minimal genome is 113 kbp long and contains 151 genes. We detail building blocks already in place and major hurdles to overcome for completion.
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  7. 7
    Schwille, P. Bottom-Up Synthetic Biology: Engineering in a Tinkerer ’ s World. Science. 2011, 333 (6047), 1252– 1254,  DOI: 10.1126/science.1211701
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    Bottom-Up Synthetic Biology: Engineering in a Tinkerer's World
    Schwille, Petra
    Science (Washington, DC, United States) (2011), 333 (6047), 1252-1254CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    A review. How synthetic can "synthetic biol." be A literal interpretation of the name of this new life science discipline invokes expectations of the systematic construction of biol. systems with cells being built module by module-from the bottom up. But can this possibly be achieved, taking into account the enormous complexity and redundancy of living systems, which distinguish them quite remarkably from design features that characterize human inventions There are several recent developments in biol., in tight conjunction with quant. disciplines, that may bring this literal perspective into the realm of the possible. However, such bottom-up engineering requires tools that were originally designed by nature's greatest tinkerer: evolution.
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  8. 8
    Caschera, F.; Noireaux, V. Integration of Biological Parts toward the Synthesis of a Minimal Cell. Curr. Opin. Chem. Biol. 2014, 22, 85– 91,  DOI: 10.1016/j.cbpa.2014.09.028
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    8
    Integration of biological parts toward the synthesis of a minimal cell
    Caschera, Filippo; Noireaux, Vincent
    Current Opinion in Chemical Biology (2014), 22 (), 85-91CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
    A review. Various approaches are taken to construct synthetic cells in the lab., a challenging goal that became exptl. imaginable over the past two decades. The construction of protocells, which explores scenarios of the origin of life, has been the original motivations for such projects. With the advent of the synthetic biol. era, bottom-up engineering approaches to synthetic cells are now conceivable. The modular design emerges as the most robust framework to construct a minimal cell from natural mol. components. Although significant advances have been made for each piece making this complex puzzle, the integration of the three fundamental parts, information-metab.-self-organization, into cell-sized liposomes capable of sustained reprodn. has failed so far. Our inability to connect these three elements is also a major limitation in this research area. New methods, such as machine learning coupled to high-throughput techniques, should be exploited to accelerate the cell-free synthesis of complex biochem. systems.
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  9. 9
    Olivi, L.; Berger, M.; Creyghton, R. N. P.; De Franceschi, N.; Dekker, C.; Mulder, B. M.; Claassens, N. J.; ten Wolde, P. R.; van der Oost, J. Towards a Synthetic Cell Cycle. Nat. Commun. 2021, 12 (1), 4531,  DOI: 10.1038/s41467-021-24772-8
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    Towards a synthetic cell cycle
    Olivi, Lorenzo; Berger, Mareike; Creyghton, Ramon N. P.; De Franceschi, Nicola; Dekker, Cees; Mulder, Bela M.; Claassens, Nico J.; ten Wolde, Pieter Rein; van der Oost, John
    Nature Communications (2021), 12 (1), 4531CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Abstr.: Recent developments in synthetic biol. may bring the bottom-up generation of a synthetic cell within reach. A key feature of a living synthetic cell is a functional cell cycle, in which DNA replication and segregation as well as cell growth and division are well integrated. Here, we describe different approaches to recreate these processes in a synthetic cell, based on natural systems and/or synthetic alternatives. Although some individual machineries have recently been established, their integration and control in a synthetic cell cycle remain to be addressed. In this Perspective, we discuss potential paths towards an integrated synthetic cell cycle.
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  10. 10
    Pols, T.; Sikkema, H. R.; Gaastra, B. F.; Frallicciardi, J.; Śmigiel, W. M.; Singh, S.; Poolman, B. A Synthetic Metabolic Network for Physicochemical Homeostasis. Nat. Commun. 2019, 10 (1), 1– 13,  DOI: 10.1038/s41467-019-12287-2
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    10
    A synthetic metabolic network for physicochemical homeostasis
    Pols, Tjeerd; Sikkema, Hendrik R.; Gaastra, Bauke F.; Frallicciardi, Jacopo; Smigiel, Wojciech M.; Singh, Shubham; Poolman, Bert
    Nature Communications (2019), 10 (1), 1-13CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    One of the grand challenges in chem. is the construction of functional out-of-equil. networks, which are typical of living cells. Building such a system from mol. components requires control over the formation and degrdn. of the interacting chems. and homeostasis of the internal phys.-chem. conditions. The provision and consumption of ATP lies at the heart of this challenge. Here we report the in vitro construction of a pathway in vesicles for sustained ATP prodn. that is maintained away from equil. by control of energy dissipation. We maintain a const. level of ATP with varying load on the system. The pathway enables us to control the transmembrane fluxes of osmolytes and to demonstrate basic physicochem. homeostasis. Our work demonstrates metabolic energy conservation and cell vol. regulatory mechanisms in a cell-like system at a level of complexity minimally needed for life.
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  11. 11
    Sikkema, H. R.; Gaastra, B. F.; Pols, T.; Poolman, B. Cell Fuelling and Metabolic Energy Conservation in Synthetic Cells. ChemBioChem. 2019, 20 (20), 2581– 2592,  DOI: 10.1002/cbic.201900398
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    11
    Cell Fuelling and Metabolic Energy Conservation in Synthetic Cells
    Sikkema, Hendrik R.; Gaastra, Bauke F.; Pols, Tjeerd; Poolman, Bert
    ChemBioChem (2019), 20 (20), 2581-2592CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. We are aiming for a blue print for synthesizing (moderately complex) subcellular systems from mol. components and ultimately for constructing life. However, without comprehensive instructions and design principles, we rely on simple reaction routes to operate the essential functions of life. The first forms of synthetic life will not make every building block for polymers de novo according to complex pathways, rather they will be fed with amino acids, fatty acids and nucleotides. Controlled energy supply is crucial for any synthetic cell, no matter how complex. Herein, we describe the simplest pathways for the efficient generation of ATP and electrochem. ion gradients. We have estd. the demand for ATP by polymer synthesis and maintenance processes in small cell-like systems, and we describe circuits to control the need for ATP. We also present fluorescence-based sensors for pH, ionic strength, excluded vol., ATP/ADP, and viscosity, which allow the major physicochem. conditions inside cells to be monitored and tuned.
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  12. 12
    Bailoni, E.; Poolman, B. ATP Recycling Fuels Sustainable Glycerol 3 - Phosphate Formation in Synthetic Cells Fed by Dynamic Dialysis. ACS Synth. Biol. 2022, 11 (7), 2348– 2360,  DOI: 10.1021/acssynbio.2c00075
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    12
    ATP Recycling Fuels Sustainable Glycerol 3-Phosphate Formation in Synthetic Cells Fed by Dynamic Dialysis
    Bailoni, Eleonora; Poolman, Bert
    ACS Synthetic Biology (2022), 11 (7), 2348-2360CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    The bottom-up construction of an autonomously growing, self-reproducing cell represents a great challenge for synthetic biol. Synthetic cellular systems are envisioned as out-of-equil. enzymic networks encompassed by a selectively open phospholipid bilayer allowing for protein-mediated communication; internal metabolite recycling is another key aspect of a sustainable metab. Importantly, gaining tight control over the external medium is essential to avoid thermodn. equil. due to nutrient depletion or waste buildup in a closed compartment (e.g., a test tube). Implementing a sustainable strategy for phospholipid biosynthesis is key to expanding the cellular boundaries. However, phospholipid biosynthesis is currently limited by substrate availability, e.g., of glycerol 3-phosphate, the essential core of phospholipid headgroups. Here, we reconstitute an enzymic network for sustainable glycerol 3-phosphate synthesis inside large unilamellar vesicles. We exploit the Escherichia coli glycerol kinase GlpK to synthesize glycerol 3-phosphate from externally supplied glycerol. We fuel phospholipid headgroup formation by sustainable L-arginine breakdown. In addn., we design and characterize a dynamic dialysis setup optimized for synthetic cells, which is used to control the external medium compn. and to achieve sustainable glycerol 3-phosphate synthesis.
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  13. 13
    Lee, K. Y.; Park, S. J.; Lee, K. A.; Kim, S. H.; Kim, H.; Meroz, Y.; Mahadevan, L.; Jung, K. H.; Ahn, T. K.; Parker, K. K.; Shin, K. Photosynthetic Artificial Organelles Sustain and Control ATP-Dependent Reactions in a Protocellular System. Nat. Biotechnol. 2018, 36 (6), 530– 535,  DOI: 10.1038/nbt.4140
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    13
    Photosynthetic artificial organelles sustain and control ATP-dependent reactions in a protocellular system
    Lee, Keel Yong; Park, Sung-Jin; Lee, Keon Ah; Kim, Se-Hwan; Kim, Heeyeon; Meroz, Yasmine; Mahadevan, L.; Jung, Kwang-Hwan; Ahn, Tae Kyu; Parker, Kevin Kit; Shin, Kwanwoo
    Nature Biotechnology (2018), 36 (6), 530-535CODEN: NABIF9; ISSN:1087-0156. (Nature Research)
    Inside cells, complex metabolic reactions are distributed across the modular compartments of organelles. Reactions in organelles have been recapitulated in vitro by reconstituting functional protein machineries into membrane systems. However, maintaining and controlling these reactions is challenging. Here we designed, built, and tested a switchable, light-harvesting organelle that provides both a sustainable energy source and a means of directing intravesicular reactions. An ATP (ATP) synthase and two photoconverters (plant-derived photosystem II and bacteria-derived proteorhodopsin) enable ATP synthesis. Independent optical activation of the two photoconverters allows dynamic control of ATP synthesis: red light facilitates and green light impedes ATP synthesis. We encapsulated the photosynthetic organelles in a giant vesicle to form a protocellular system and demonstrated optical control of two ATP-dependent reactions, carbon fixation and actin polymn., with the latter altering outer vesicle morphol. Switchable photosynthetic organelles may enable the development of biomimetic vesicle systems with regulatory networks that exhibit homeostasis and complex cellular behaviors.
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    Partipilo, M.; Ewins, E. J.; Frallicciardi, J.; Robinson, T.; Poolman, B.; Slotboom, D. J. Minimal Pathway for the Regeneration of Redox Cofactors. JACS Au 2021, 1 (12), 2280– 2293,  DOI: 10.1021/jacsau.1c00406
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    14
    Minimal Pathway for the Regeneration of Redox Cofactors
    Partipilo, Michele; Ewins, Eleanor J.; Frallicciardi, Jacopo; Robinson, Tom; Poolman, Bert; Slotboom, Dirk Jan
    JACS Au (2021), 1 (12), 2280-2293CODEN: JAAUCR; ISSN:2691-3704. (American Chemical Society)
    Effective metabolic pathways are essential for the construction of in vitro systems mimicking the biochem. complexity of living cells. Such pathways require the inclusion of a metabolic branch that ensures the availability of reducing equiv. Here, we built a minimal enzymic pathway confinable in the lumen of liposomes, in which the redox status of the nicotinamide cofactors NADH and NADPH is controlled by an externally provided formate. Formic acid permeates the membrane where a luminal formate dehydrogenase uses NAD+ to form NADH and carbon dioxide. Carbon dioxide diffuses out of the liposomes, leaving only the reducing equiv. in the lumen. A sol. transhydrogenase subsequently utilizes NADH for redn. of NADP+ thereby making NAD+ available again for the first reaction. The pathway is functional in liposomes ranging from a few hundred nanometers in diam. (large unilamellar vesicles) up to several tens of micrometers (giant unilamellar vesicles) and remains active over a period of 7 days. We demonstrate that the downstream biochem. process of redn. of glutathione disulfide can be driven by the transfer of reducing equiv. from formate via NAD(P)H, thereby providing a versatile set of electron donors for reductive metab.
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    Camps, M.; Herman, A.; Loh, E.; Loeb, L. A. Genetic Constraints on Protein Evolution. Crit. Rev. Biochem. Mol. Biol. 2007, 42 (5), 313– 326,  DOI: 10.1080/10409230701597642
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    Genetic constraints on protein evolution
    Camps, Manel; Herman, Asael; Loh, Ern; Loeb, Lawrence A.
    Critical Reviews in Biochemistry and Molecular Biology (2007), 42 (5), 313-326CODEN: CRBBEJ; ISSN:1040-9238. (Informa Healthcare)
    A review. Evolution requires the generation and optimization of new traits ("adaptation") and involves the selection of mutations that improve cellular function. These mutations are assumed to arise by selection of neutral mutations present at all times in the population. Here, the authors review recent evidence that indicates that deleterious mutations are more frequent in the population than previously recognized and that these mutations play a significant role in protein evolution through continuous pos. selection. Pos. selected mutations include adaptive mutations, i.e., mutations that directly affect enzymic function, and compensatory mutations, which suppress the pleiotropic effects of adaptive mutations. Compensatory mutations are by far the most frequent of the 2 and would allow potentially adaptive but deleterious mutations to persist long enough in the population to be pos. selected during episodes of adaptation. Compensatory mutations are, by definition, context-dependent and thus constrain the paths available for evolution. This provides a mechanistic basis for the examples of highly constrained evolutionary landscapes and parallel evolution reported in natural and exptl. populations. The present review article describes these recent advances in the field of protein evolution and discusses their implications for understanding the genetic basis of disease and for protein engineering in vitro.
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    Coombes, D.; Moir, J. W. B.; Poole, A. M.; Cooper, T. F.; Dobson, R. C. J. The Fitness Challenge of Studying Molecular Adaptation. Biochem. Soc. Trans. 2019, 47 (5), 1533– 1542,  DOI: 10.1042/BST20180626
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    The fitness challenge of studying molecular adaptation
    Coombes, David; Moir, James W. B.; Poole, Anthony M.; Cooper, Tim F.; Dobson, Renwick C. J.
    Biochemical Society Transactions (2019), 47 (5), 1533-1542CODEN: BCSTB5; ISSN:0300-5127. (Portland Press Ltd.)
    A review. Advances in bioinformatics and high-throughput genetic anal. increasingly allow us to predict the genetic basis of adaptive traits. These predictions can be tested and confirmed, but the mol.-level changes - i.e. the mol. adaptation - that link genetic differences to organism fitness remain generally unknown. In recent years, a series of studies have started to unpick the mechanisms of adaptation at the mol. level. In particular, this work has examd. how changes in protein function, activity, and regulation cause improved organismal fitness. Key to addressing mol. adaptations is identifying systems and designing expts. that integrate changes in the genome, protein chem. (mol. phenotype), and fitness. Knowledge of the mol. changes underpinning adaptations allow new insight into the constraints on, and repeatability of adaptations, and of the basis of non-additive interactions between adaptive mutations. Here we critically discuss a series of studies that examine the mol.-level adaptations that connect genetic changes and fitness.
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    Schmidt, S.; Sunyaev, S.; Bork, P.; Dandekar, T. Metabolites: A Helping Hand for Pathway Evolution?. Trends Biochem. Sci. 2003, 28 (6), 336– 341,  DOI: 10.1016/S0968-0004(03)00114-2
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    Metabolites: a helping hand for pathway evolution?
    Schmidt, Steffen; Sunyaev, Shamil; Bork, Peer; Dandekar, Thomas
    Trends in Biochemical Sciences (2003), 28 (6), 336-341CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Science Ltd.)
    A review. The evolution of enzymes and pathways is under debate. Recent studies show that recruitment of single enzymes from different pathways could be the driving force for pathway evolution. Other mechanisms of evolution, such as pathway duplication, enzyme specialization, de novo invention of pathways or retro-evolution of pathways, appear to be less abundant. Twenty percent of enzyme superfamilies are quite variable, not only in changing reaction chem. or metabolite type but in changing both at the same time. These variable superfamilies account for nearly half of all known reactions. The most frequently occurring metabolites provide a helping hand for such changes because they can be accommodated by many enzyme superfamilies. Thus, a picture is emerging in which new pathways are evolving from central metabolites by preference, thereby keeping the overall topol. of the metabolic network.
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    Pfeiffer, T.; Soyer, O. S.; Bonhoeffer, S. The Evolution of Connectivity in Metabolic Networks. PLoS Biol. 2005, 3 (7), e228,  DOI: 10.1371/journal.pbio.0030228
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    Sajed, T.; Marcu, A.; Ramirez, M.; Pon, A.; Guo, A. C.; Knox, C.; Wilson, M.; Grant, J. R.; Djoumbou, Y.; Wishart, D. S. ECMDB 2.0: A Richer Resource for Understanding the Biochemistry of E. Coli. Nucleic Acids Res. 2016, 44 (D1), D495– D501,  DOI: 10.1093/nar/gkv1060
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    19
    ECMDB 2.0: a richer resource for understanding the biochemistry of E. coli
    Sajed, Tanvir; Marcu, Ana; Ramirez, Miguel; Pon, Allison; Guo, An Chi; Knox, Craig; Wilson, Michael; Grant, Jason R.; Djoumbou, Yannick; Wishart, David S.
    Nucleic Acids Research (2016), 44 (D1), D495-D501CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    ECMDB or the Escherichia coli metabolome database is a comprehensive database contg. detailed information about the genome and metabolome of E. coli (K-12). First released in 2012, the ECMDB has undergone substantial expansion and many modifications over the past 4 years. This manuscript describes the most recent version of ECMDB (ECMDB 2.0). In particular, it provides a comprehensive update of the database that was previously described in the 2013 NAR Database Issue and details many of the addns. and improvements made to the ECMDB over that time. Some of the most important or significant enhancements include a 13-fold increase in the no. of metabolic pathway diagrams (from 125 to 1650), a 3-fold increase in the no. of compds. linked to pathways (from 1058 to 3280), the addn. of dozens of operon/metabolite signaling pathways, a 44% increase in the no. of compds. in the database (from 2610 to 3760), a 7-fold increase in the no. of compds. with NMR or MS spectra (from 412 to 3261) and a massive increase in the no. of external links to other E. coli or chem. resources. These addns., along with many other enhancements aimed at improving the ease or speed of querying, searching and viewing the data within ECMDB should greatly facilitate the understanding of not only the metab. of E. coli, but also allow the in-depth exploration of its extensive metabolic networks, its many signaling pathways and its essential biochem.
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  20. 20
    Blank, L. M.; Ebert, B. E.; Buehler, K.; Bühler, B. Redox Biocatalysis and Metabolism: Molecular Mechanisms and Metabolic Network Analysis. Antioxidants Redox Signal. 2010, 13 (3), 349– 394,  DOI: 10.1089/ars.2009.2931
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    20
    Redox biocatalysis and metabolism: molecular mechanisms and metabolic network analysis
    Blank Lars M; Ebert Birgitta E; Buehler Katja; Buhler Bruno
    Antioxidants & redox signaling (2010), 13 (3), 349-94 ISSN:.
    Whole-cell biocatalysis utilizes native or recombinant enzymes produced by cellular metabolism to perform synthetically interesting reactions. Besides hydrolases, oxidoreductases represent the most applied enzyme class in industry. Oxidoreductases are attributed a high future potential, especially for applications in the chemical and pharmaceutical industries, as they enable highly interesting chemistry (e.g., the selective oxyfunctionalization of unactivated C-H bonds). Redox reactions are characterized by electron transfer steps that often depend on redox cofactors as additional substrates. Their regeneration typically is accomplished via the metabolism of whole-cell catalysts. Traditionally, studies towards productive redox biocatalysis focused on the biocatalytic enzyme, its activity, selectivity, and specificity, and several successful examples of such processes are running commercially. However, redox cofactor regeneration by host metabolism was hardly considered for the optimization of biocatalytic rate, yield, and/or titer. This article reviews molecular mechanisms of oxidoreductases with synthetic potential and the host redox metabolism that fuels biocatalytic reactions with redox equivalents. The tools discussed in this review for investigating redox metabolism provide the basis for studies aiming at a deeper understanding of the interplay between synthetically active enzymes and metabolic networks. The ultimate goal of rational whole-cell biocatalyst engineering and use for fine chemical production is discussed.
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  21. 21
    Agledal, L.; Niere, M.; Ziegler, M. The Phosphate Makes a Difference: Cellular Functions of NADP. Redox Rep. 2010, 15 (1), 2– 10,  DOI: 10.1179/174329210X12650506623122
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    21
    The phosphate makes a difference: cellular functions of NADP
    Agledal, Line; Niere, Marc; Ziegler, Mathias
    Redox Report (2010), 15 (1), 2-10CODEN: RDRPE4; ISSN:1351-0002. (Maney Publishing)
    A review. Recent research has unraveled a no. of unexpected functions of the pyridine nucleotides. In this review, we will highlight the variety of known physiol. roles of NADP. In its reduced form (NADPH), this mol. represents a universal electron donor, not only to drive biosynthetic pathways. Perhaps even more importantly, NADPH is the unique provider of reducing equiv. to maintain or regenerate the cellular detoxifying and antioxidative defense systems. The roles of NADPH in redox sensing and as substrate for NADPH oxidases to generate reactive oxygen species further extend its scope of functions. NADP+, on the other hand, has acquired signaling functions. Its conversion to second messengers in calcium signaling may have crit. impact on important cellular processes. The generation of NADP by NAD kinases is a key determinant of the cellular NADP concn. The regulation of these enzymes may, therefore, be crit. to feed the diversity of NADP-dependent processes adequately. The increasing recognition of the multiple roles of NADP has thus led to exciting new insights in this expanding field.
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  22. 22
    Spaans, S. K.; Weusthuis, R. A.; van der Oost, J.; Kengen, S. W. M. NADPH-Generating Systems in Bacteria and Archaea. Front. Microbiol. 2015, 6, 742,  DOI: 10.3389/fmicb.2015.00742
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    22
    NADPH-generating systems in bacteria and archaea
    Spaans Sebastiaan K; van der Oost John; Kengen Serve W M; Weusthuis Ruud A
    Frontiers in microbiology (2015), 6 (), 742 ISSN:1664-302X.
    Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components and many products in biotechnology. The efficient synthesis of many of these products, however, is limited by the rate of NADPH regeneration. Hence, a thorough understanding of the reactions involved in the generation of NADPH is required to increase its turnover through rational strain improvement. Traditionally, the main engineering targets for increasing NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA) cycle. However, the importance of alternative NADPH-generating reactions has recently become evident. In the current review, the major canonical and non-canonical reactions involved in the production and regeneration of NADPH in prokaryotes are described, and their key enzymes are discussed. In addition, an overview of how different enzymes have been applied to increase NADPH availability and thereby enhance productivity is provided.
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    Fuhrer, T.; Sauer, U. Different Biochemical Mechanisms Ensure Network-Wide Balancing of Reducing Equivalents in Microbial Metabolism. J. Bacteriol. 2009, 191 (7), 2112– 2121,  DOI: 10.1128/JB.01523-08
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    23
    Different biochemical mechanisms ensure network-wide balancing of reducing equivalents in microbial metabolism
    Fuhrer, Tobias; Sauer, Uwe
    Journal of Bacteriology (2009), 191 (7), 2112-2121CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
    To sustain growth, the catabolic formation of the redox equiv. NADPH must be balanced with the anabolic demand. The mechanisms that ensure such network-wide balancing, however, are presently not understood. Based on 13C-detected intracellular fluxes, metabolite concns., and cofactor specificities for all relevant central metabolic enzymes, we have quantified catabolic NADPH prodn. in Agrobacterium tumefaciens, Bacillus subtilis, Escherichia coli, Paracoccus versutus, Pseudomonas fluorescens, Rhodobacter sphaeroides, Sinorhizobium meliloti, and Zymomonas mobilis. For six species, the estd. NADPH prodn. from glucose catabolism exceeded the requirements for biomass synthesis. Exceptions were P. fluorescens, with balanced rates, and E. coli, with insufficient catabolic prodn., in which about one-third of the NADPH is supplied via the membrane-bound transhydrogenase PntAB. P. versutus and B. subtilis were the only species that appear to rely on transhydrogenases for balancing NADPH overprodn. during growth on glucose. In the other four species, the main but not exclusive redox-balancing mechanism appears to be the dual cofactor specificities of several catabolic enzymes and/or the existence of isoenzymes with distinct cofactor specificities, in particular glucose 6-phosphate dehydrogenase. An unexpected key finding for all species, except E. coli and B. subtilis, was the lack of cofactor specificity in the oxidative pentose phosphate pathway, which contrasts with the textbook view of the pentose phosphate pathway dehydrogenases as being NADP+ dependent.
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  24. 24
    Goldford, J. E.; George, A. B.; Flamholz, A. I.; Segre, D. Protein Cost Minimization Promotes the Emergence of Coenzyme Redundancy. Proc. Natl. Acad. Sci. U. S. A. 2022, 119 (14), e2110787119,  DOI: 10.1073/pnas.2110787119
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    24
    Protein cost minimization promotes the emergence of coenzyme redundancy
    Goldford, Joshua E.; George, Ashish B.; Flamholz, Avi I.; Segre, Daniel
    Proceedings of the National Academy of Sciences of the United States of America (2022), 119 (14), e2110787119CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Coenzymes distribute a variety of chem. moieties throughout cellular metab., participating in group (e.g., phosphate and acyl) and electron transfer. For a variety of reactions requiring acceptors or donors of specific resources, there often exist degenerate sets of mols. [e.g., NAD(H) and NADP(H)] that carry out similar functions. Although the physiol. roles of various coenzyme systems are well established, it is unclear what selective pressures may have driven the emergence of coenzyme redundancy. Here, we use genome-wide metabolic modeling approaches to decomp. the selective pressures driving enzymic specificity for either NAD(H) or NADP(H) in the metabolic network of Escherichia coli. We found that few enzymes are thermodynamically constrained to using a single coenzyme, and in principle a metabolic network relying on only NAD(H) is feasible. However, structural and sequence analyses revealed widespread conservation of residues that retain selectivity for either NAD(H) or NADP(H), suggesting that addnl. forces may shape specificity. Using a model accounting for the cost of oxidoreductase enzyme expression, we found that coenzyme redundancy universally reduces the minimal amt. of protein required to catalyze coenzyme-coupled reactions, inducing individual reactions to strongly prefer one coenzyme over another when reactions are near thermodn. equil. We propose that protein minimization generically promotes coenzyme redundancy and that coenzymes typically thought to exist in a single pool (e.g., CoA [CoA]) may exist in more than one form (e.g., dephospho-CoA).
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    Xiao, W.; Wang, R. S.; Handy, D. E.; Loscalzo, J. NAD(H) and NADP(H) Redox Couples and Cellular Energy Metabolism. Antioxidants Redox Signal. 2018, 28 (3), 251– 272,  DOI: 10.1089/ars.2017.7216
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    25
    NAD(H) and NADP(H) Redox Couples and Cellular Energy Metabolism
    Xiao Wusheng; Wang Rui-Sheng; Handy Diane E; Loscalzo Joseph
    Antioxidants & redox signaling (2018), 28 (3), 251-272 ISSN:.
    SIGNIFICANCE: The nicotinamide adenine dinucleotide (NAD(+))/reduced NAD(+) (NADH) and NADP(+)/reduced NADP(+) (NADPH) redox couples are essential for maintaining cellular redox homeostasis and for modulating numerous biological events, including cellular metabolism. Deficiency or imbalance of these two redox couples has been associated with many pathological disorders. Recent Advances: Newly identified biosynthetic enzymes and newly developed genetically encoded biosensors enable us to understand better how cells maintain compartmentalized NAD(H) and NADP(H) pools. The concept of redox stress (oxidative and reductive stress) reflected by changes in NAD(H)/NADP(H) has increasingly gained attention. The emerging roles of NAD(+)-consuming proteins in regulating cellular redox and metabolic homeostasis are active research topics. CRITICAL ISSUES: The biosynthesis and distribution of cellular NAD(H) and NADP(H) are highly compartmentalized. It is critical to understand how cells maintain the steady levels of these redox couple pools to ensure their normal functions and simultaneously avoid inducing redox stress. In addition, it is essential to understand how NAD(H)- and NADP(H)-utilizing enzymes interact with other signaling pathways, such as those regulated by hypoxia-inducible factor, to maintain cellular redox homeostasis and energy metabolism. FUTURE DIRECTIONS: Additional studies are needed to investigate the inter-relationships among compartmentalized NAD(H)/NADP(H) pools and how these two dinucleotide redox couples collaboratively regulate cellular redox states and cellular metabolism under normal and pathological conditions. Furthermore, recent studies suggest the utility of using pharmacological interventions or nutrient-based bioactive NAD(+) precursors as therapeutic interventions for metabolic diseases. Thus, a better understanding of the cellular functions of NAD(H) and NADP(H) may facilitate efforts to address a host of pathological disorders effectively. Antioxid. Redox Signal. 28, 251-272.
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  26. 26
    Breuer, M.; Earnest, T. M.; Merryman, C.; Wise, K. S.; Sun, L.; Lynott, M. R.; Hutchison, C. A.; Smith, H. O.; Lapek, J. D.; Gonzalez, D. J.; Crécy-Lagard, V. de; Haas, D.; Hanson, A. D.; Labhsetwar, P.; Glass, J. I.; Luthey-Schulten, Z. Essential Metabolism for a Minimal Cell. eLife 2019, 8, e36842,  DOI: 10.7554/eLife.36842
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    Wichmann, R.; Vasic-Racki, D. Cofactor Regeneration at the Lab Scale. Adv. Biochem. Eng. Biotechnol. 2005, 92, 225– 260,  DOI: 10.1007/b98911
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    Cofactor regeneration at the lab scale
    Wichmann, R.; Vasic-Racki, D.
    Advances in Biochemical Engineering/Biotechnology (2005), 92 (Technology Transfer in Biotechnology), 225-260CODEN: ABEBDZ; ISSN:0724-6145. (Springer GmbH)
    A review. Progress made in lab.-scale applications of various coenzyme regeneration systems over the last two decades has mainly focused on the applications of NAD+/NADH- and NADP+/NADPH-dependent oxidoreductase reactions. In situ regeneration systems for these reactions, as well as whole cell, enzymic, electro-enzymic, chem., and photochem. reactions are presented, including details about their efficiency and novelty. The progress of enzyme reaction engineering is also reported.
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    Weckbecker, A.; Gröger, H.; Hummel, W. Regeneration of Nicotinamide Coenzymes: Principles and Applications for the Synthesis of Chiral Compounds. Biosyst. Eng. I 2010, 195– 242,  DOI: 10.1007/10_2009_55
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    Shi, T.; Han, P.; You, C.; Zhang, Y. H. P. J. An in Vitro Synthetic Biology Platform for Emerging Industrial Biomanufacturing: Bottom-up Pathway Design. Synth. Syst. Biotechnol. 2018, 3 (3), 186– 195,  DOI: 10.1016/j.synbio.2018.05.002
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    An in vitro synthetic biology platform for emerging industrial biomanufacturing: Bottom-up pathway design
    Shi Ting; Han Pingping; You Chun; Zhang Yi-Heng P Job
    Synthetic and systems biotechnology (2018), 3 (3), 186-195 ISSN:.
    Although most in vitro (cell-free) synthetic biology projects are usually used for the purposes of fundamental research or the formation of high-value products, in vitro synthetic biology platform, which can implement complicated biochemical reactions by the in vitro assembly of numerous enzymes and coenzymes, has been proposed for low-cost biomanufacturing of bioenergy, food, biochemicals, and nutraceuticals. In addition to the most important advantage-high product yield, in vitro synthetic biology platform features several other biomanufacturing advantages, such as fast reaction rate, easy product separation, open process control, broad reaction condition, tolerance to toxic substrates or products, and so on. In this article, we present the basic bottom-up design principles of in vitro synthetic pathway from basic building blocks-BioBricks (thermoenzymes and/or immobilized enzymes) to building modules (e.g., enzyme complexes or multiple enzymes as a module) with specific functions. With development in thermostable building blocks-BioBricks and modules, the in vitro synthetic biology platform would open a new biomanufacturing age for the cost-competitive production of biocommodities.
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    Tassano, E.; Hall, M. Enzymatic Self-Sufficient Hydride Transfer Processes. Chem. Soc. Rev. 2019, 48 (23), 5596– 5615,  DOI: 10.1039/C8CS00903A
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    Enzymatic self-sufficient hydride transfer processes
    Tassano, Erika; Hall, Melanie
    Chemical Society Reviews (2019), 48 (23), 5596-5615CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review. A no. of self-sufficient hydride transfer processes have been reported in biocatalysis, with a common feature being the dependence on nicotinamide as a cofactor. This cofactor is provided in catalytic amts. and serves as a hydride shuttle to connect two or more enzymic redox events, usually ensuring overall redox neutrality. Creative systems were designed to produce synthetic sequences characterized by high hydride economy, typically going in hand with excellent atom economy. Several redox enzymes have been successfully combined in one-pot one-step to allow functionalization of a large variety of mols. while preventing byproduct formation. This review analyzes and classifies the various strategies, with a strong focus on efficiency, which is evaluated here in terms of the hydride economy and measured by the turnover no. of the nicotinamide cofactor(s). The review ends with a crit. evaluation of the reported systems and highlights areas where further improvements might be desirable.
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    Dekker, J. P.; Boekema, E. J. Supramolecular Organization of Thylakoid Membrane Proteins in Green Plants. Biochim. Biophys. Acta - Bioenerg. 2005, 1706 (1–2), 12– 39,  DOI: 10.1016/j.bbabio.2004.09.009
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    Supramolecular organization of thylakoid membrane proteins in green plants
    Dekker, Jan P.; Boekema, Egbert J.
    Biochimica et Biophysica Acta, Bioenergetics (2005), 1706 (1-2), 12-39CODEN: BBBEB4; ISSN:0005-2728. (Elsevier B.V.)
    A review. The light reactions of photosynthesis in green plants are mediated by four large protein complexes, embedded in the thylakoid membrane of the chloroplast. Photosystem I (PSI) and Photosystem II (PSII) are both organized into large supercomplexes with variable amts. of membrane-bound peripheral antenna complexes. PSI consists of a monomeric core complex with single copies of four different LHCI proteins and has binding sites for addnl. LHCI and/or LHCII complexes. PSII supercomplexes are dimeric and contain usually two to four copies of trimeric LHCII complexes. These supercomplexes have a further tendency to assoc. into megacomplexes or into cryst. domains, of which several types have been characterized. Together with the specific lipid compn., the structural features of the main protein complexes of the thylakoid membranes form the main trigger for the segregation of PSII and LHCII from PSI and ATPase into stacked grana membranes. We suggest that the margins, the strongly folded regions of the membranes that connect the grana, are essentially protein-free, and that protein-protein interactions in the lumen also det. the shape of the grana. We also discuss which mechanisms det. the stacking of the thylakoid membranes and how the supramol. organization of the pigment-protein complexes in the thylakoid membrane and their flexibility may play roles in various regulatory mechanisms of green plant photosynthesis.
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    Thauer, R. K.; Jungermann, K.; Decker, K. Energy Conservation in Chemotrophic Anaerobic Bacteria. Bacteriol. Rev. 1977, 41 (1), 100– 180,  DOI: 10.1128/br.41.1.100-180.1977
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    Energy conservation in chemotrophic anaerobic bacteria
    Thauer, Rudolf K.; Jungermann, Kurt; Decker, Karl
    Bacteriological Reviews (1977), 41 (1), 100-80CODEN: BAREA8; ISSN:0005-3678.
    A review with 743 refs.
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    Lehninger, A. L.; Lehninger Principles of Biochemistry; Macmillan, 2005.
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    Relyea, H. A.; Van Der Donk, W. A. Mechanism and Applications of Phosphite Dehydrogenase. Bioorg. Chem. 2005, 33 (3), 171– 189,  DOI: 10.1016/j.bioorg.2005.01.003
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    Mechanism and applications of phosphite dehydrogenase
    Relyea, Heather A.; van der Donk, Wilfred A.
    Bioorganic Chemistry (2005), 33 (3), 171-189CODEN: BOCMBM; ISSN:0045-2068. (Elsevier)
    A review. Phosphite dehydrogenase catalyzes the NAD+-dependent oxidn. of hydrogen phosphonate (common name phosphite) to phosphate in what amts. to a formal phosphoryl transfer reaction from hydride to hydroxide. This review places the enzyme in the context of phosphorus redox metab. in nature and discusses the results of mechanistic investigations into its reaction mechanism. The potential of the enzyme as a NAD(P)H cofactor regeneration system is discussed as well as efforts to engineer the cofactor specificity of the protein.
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    Wu, J.; Huang, Y.; Ye, W.; Li, Y. CO2 Reduction: From the Electrochemical to Photochemical Approach. Adv. Sci. 2017, 4 (11), 1700194,  DOI: 10.1002/advs.201700194
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    CO2 Reduction: From the Electrochemical to Photochemical Approach
    Wu Jinghua; Huang Yang; Ye Wen; Li Yanguang
    Advanced science (Weinheim, Baden-Wurttemberg, Germany) (2017), 4 (11), 1700194 ISSN:2198-3844.
    Increasing CO2 concentration in the atmosphere is believed to have a profound impact on the global climate. To reverse the impact would necessitate not only curbing the reliance on fossil fuels but also developing effective strategies capture and utilize CO2 from the atmosphere. Among several available strategies, CO2 reduction via the electrochemical or photochemical approach is particularly attractive since the required energy input can be potentially supplied from renewable sources such as solar energy. In this Review, an overview on these two different but inherently connected approaches is provided and recent progress on the development, engineering, and understanding of CO2 reduction electrocatalysts and photocatalysts is summarized. First, the basic principles that govern electrocatalytic or photocatalytic CO2 reduction and their important performance metrics are discussed. Then, a detailed discussion on different CO2 reduction electrocatalysts and photocatalysts as well as their generally designing strategies is provided. At the end of this Review, perspectives on the opportunities and possible directions for future development of this field are presented.
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    Beber, M. E.; Gollub, M. G.; Mozaffari, D.; Shebek, K. M.; Flamholz, A. I.; Milo, R.; Noor, E. EQuilibrator 3.0: A Database Solution for Thermodynamic Constant Estimation. Nucleic Acids Res. 2022, 50 (D1), D603– D609,  DOI: 10.1093/nar/gkab1106
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    The eQuilibrator 3.0: a database solution for thermodynamic constant estimation
    Beber, Moritz E.; Gollub, Mattia G.; Mozaffari, Dana; Shebek, Kevin M.; Flamholz, Avi I.; Milo, Ron; Noor, Elad
    Nucleic Acids Research (2022), 50 (D1), D603-D609CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    EQuilibrator (equilibrator.weizmann.ac.il) is a database of biochem. equil. consts. and Gibbs free energies, originally designed as a web-based interface. While the website now counts around 1,000 distinct monthly users, its design could not accommodate larger compd. databases and it lacked a scalable Application Programming Interface (API) for integration into other tools developed by the systems biol. community. Here, we report on the recent updates to the database as well as the addn. of a new Python-based interface to eQuilibrator that adds many new features such as a 100-fold larger compd. database, the ability to add novel compds., improvements in speed and memory use, and correction for Mg2+ ion concns. Moreover, the new interface can compute the covariance matrix of the uncertainty between ests., for which we show the advantages and describe the application in metabolic modeling. We foresee that these improvements will make thermodn. modeling more accessible and facilitate the integration of eQuilibrator into other software platforms.
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    Cho, A.; Yun, H.; Park, J. H.; Lee, S. Y.; Park, S. Prediction of Novel Synthetic Pathways for the Production of Desired Chemicals. BMC Syst. Biol. 2010, 4 (1), 1– 16,  DOI: 10.1186/1752-0509-4-35
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    Morgado, G.; Gerngross, D.; Roberts, T. M.; Panke, S. Synthetic Biology for Cell-Free Biosynthesis: Fundamentals of Designing Novel in Vitro Multi-Enzyme Reaction Networks. Synth. Biol. Eng. 2016, 162, 117– 146,  DOI: 10.1007/10_2016_13
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    Noor, E.; Bar-Even, A.; Flamholz, A.; Reznik, E.; Liebermeister, W.; Milo, R. Pathway Thermodynamics Highlights Kinetic Obstacles in Central Metabolism. PLoS Comput. Biol. 2014, 10 (2), e1003483,  DOI: 10.1371/journal.pcbi.1003483
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    Pathway thermodynamics highlights kinetic obstacles in central metabolism
    Noor, Elad; Bar-Even, Arren; Flamholz, Avi; Reznik, Ed; Liebermeister, Wolfram; Milo, Ron
    PLoS Computational Biology (2014), 10 (2), e1003483/1-e1003483/12, 12 pp.CODEN: PCBLBG; ISSN:1553-7358. (Public Library of Science)
    In metab. research, thermodn. is usually used to det. the directionality of a reaction or the feasibility of a pathway. However, the relationship between thermodn. potentials and fluxes is not limited to questions of directionality: thermodn. also affects the kinetics of reactions through the flux-force relationship, which states that the logarithm of the ratio between the forward and reverse fluxes is directly proportional to the change in Gibbs energy due to a reaction (ΔrG'). Accordingly, if an enzyme catalyzes a reaction with a ΔrG' of -5.7 kJ/mol then the forward flux will be roughly ten times the reverse flux. As ΔrG' approaches equil. (ΔrG' = 0 kJ/mol), exponentially more enzyme counterproductively catalyzes the reverse reaction, reducing the net rate at which the reaction proceeds. Thus, the enzyme level required to achieve a given flux increases dramatically near equil. Here, we develop a framework for quantifying the degree to which pathways suffer these thermodn. limitations on flux. For each pathway, we calc. a single thermodynamically-derived metric (the Max-min Driving Force, MDF), which enables objective ranking of pathways by the degree to which their flux is constrained by low thermodn. driving force. Our framework accounts for the effect of pH, ionic strength and metabolite concn. ranges and allows us to quantify how alterations to the pathway structure affect the pathway's thermodn. Applying this methodol. to pathways of central metab. sheds light on some of their features, including metabolic bypasses (e.g., fermn. pathways bypassing substrate-level phosphorylation), substrate channeling (e.g., of oxaloacetate from malate dehydrogenase to citrate synthase), and use of alternative cofactors (e.g., quinone as an electron acceptor instead of NAD). The methods presented here place another arrow in metabolic engineers' quiver, providing a simple means of evaluating the thermodn. and kinetic quality of different pathway chemistries that produce the same mols.
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  40. 40
    Guynn, R. W.; Gelberg, H. J.; Veech, R. L. Equilibrium Constants of the Malate Dehydrogenase, Citrate Synthase, Citrate Lyase, and Acetyl Coenzyme A Hydrolysis Reactions under Physiological Conditions. J. Biol. Chem. 1973, 248 (20), 6957– 6965,  DOI: 10.1016/S0021-9258(19)43346-2
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    40
    Equilibrium constants of the malate dehydrogenase, citrate synthase, citrate lyase, and acetyl coenzyme A hydrolysis reactions under physiological conditions
    Guynn, Robert W.; Gelberg, Harris J.; Veech, Richard L.
    Journal of Biological Chemistry (1973), 248 (20), 6957-65CODEN: JBCHA3; ISSN:0021-9258.
    The obsd. equil. consts. (Kobs) of the malate dehydrogenase (EC 1.1.1.37), citrate synthase (EC 4.1.3.7), and citrate lyase (EC 4.1.3.6) reactions were detd. under near physiol. conditions (38°, pH 7.0, ionic strength 0.25, free [Mg2+] = 10-3M). From these values, the observed std. free energy change (ΔG0obs) for the hydrolysis of acetyl-CoA was detd. Under the above conditions, and taking the std. state of liq. water to have activity = unity, the equil. consts. of the 3 reactions at pH 7.0 were 2.86 × 10-5 for malate dehydrogenase, 2.24 × 106 for citrate synthase, and 2.22 M-1 for citrate lyase. The values obtained for Kobs of the citrate synthase and citrate lyase reactions vary to the same extent with the changes in Mg2+ concn. At free [Mg2+] = O, Kobs for the citrate synthase reaction is 1.01 × 106 and the Kobs for the citrate lyase reaction is 1.00M-1. In contrast, malate dehydrogenase is unaffected by the concn. of free Mg2+ up to 4 mM. From the consts. of the citrate synthase and citrate lyase reactions, the Kobs for the hydrolysis of acetyl-CoA under near physiol. conditions is calcd. to be 1.01 × 106M, corresponding to a free energy change of -8.54 kcal/mole (-35.75 kJ/mole) independent of the free Mg2+ concn.
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  41. 41
    Williamson, D. H.; Lund, P.; K, H. A. The Redox State of Nicotinamide Adenine Dinucleotide in the Cytoplasm and Mitochondria of Rat Liver. Biochem. J. 1967, 103 (2), 514,  DOI: 10.1042/bj1030514
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    41
    Redox state of free nicotinamide adenine dinucleotide in the cytoplasm and mitochondria of rat liver
    Williamson, Dermot Hedley; Lund, Patricia; Krebs, Hans A.
    Biochemical Journal (1967), 103 (), 514-27CODEN: BIJOAK; ISSN:0264-6021.
    The concns. of the oxidized and reduced substrates of the lactate, β-hydroxybutyrate, and glutamate dehydrogenase systems were measured in rat livers freeze-clamped as soon as possible after death. The substrates of these dehydrogenases are likely to be in equil. with free NAD and NADH, and the ratio of the free dinucleotides can be calcd. from the measured concns. of the substrates and the equil. consts. (Holzer, et al., CA 51, 6776h; Bucher and Klingenberg, CA 53, 22147a). The lactate dehydrogenase system reflects the NAD-to-NADH ratio in the cytoplasm, the β-hydroxybutyrate dehydrogenase that in the mitochondrial cristae, and the glutamate dehydrogenase that in the mitochondrial matrix. The equil. consts. of lactate dehydrogenase (EC 1.1.1.27), β-hydroxybutyrate dehydrogenase (EC 1.1.1.30), and malate dehydrogenase (EC 1.1.1.37) were redetd. for near-physiol. conditions (38°; ionic strength 0.25). The mean NAD-to-NADH ratio of rat liver cytoplasm was calcd. as 725 (pH 7.0) in well fed rats, 528 in starved rats, and 208 in alloxan-diabetic rats. The NAD to-NADH ratio for the mitochondrial matrix and cristae gave virtually identical values in the same metabolic state. This indicates that β-hydroxybutyrate dehydrogenase and glutamate dehydrogenase share a common pool of dinucleotide. The mean NAD-to-NADH ratio within the liver mitochondria of well fed rats was ∼8. It fell to ∼5 in starvation and rose to ∼10 in alloxan-diabetes. The NAD-to-NADH ratios of cytoplasm and mitochondria are thus greatly different and do not necessarily move in parallel when the metabolic state of the liver changes. The ratios found for the free dinucleotides differ greatly from those recorded for the total dinucleotides because much more NADH than NAD is protein bound. The bearing of these findings on various problems, including the following, is discussed: the no. of NAD-NADH pools in liver cells; the applicability of the method to tissues other than liver; the transhydrogenase activity of glutamate dehydrogenase; the physiol. significance of the difference of the redox states of mitochondria and cytoplasm; aspects of the regulation of the redox state of cell compartments; the steady-state concn. of mitochondrial oxalacetate; the relations between the redox state of cell compartments and ketosis.
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  42. 42
    Bailoni, E.; Partipilo, M.; Coenradij, J.; Grundel, D. A. J.; Slotboom, D. J.; Poolman, B. Minimal Out-of-Equilibrium Metabolism for Synthetic Cells: A Membrane Perspective. ACS Synth. Biol. 2023,  DOI: 10.1021/acssynbio.3c00062
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    Crnković, A.; Srnko, M.; Anderluh, G. Biological Nanopores: Engineering on Demand. Life 2021, 11 (1), 27,  DOI: 10.3390/life11010027
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    Biological nanopores: engineering on demand
    Crnkovic, Ana; Srnko, Marija; Anderluh, Gregor
    Life (Basel, Switzerland) (2021), 11 (1), 27CODEN: LBSIB7; ISSN:2075-1729. (MDPI AG)
    Nanopore-based sensing is a powerful technique for the detection of diverse org. and inorg. mols., long-read sequencing of nucleic acids, and single-mol. analyses of enzymic reactions. Selected from natural sources, protein-based nanopores enable rapid, label-free detection of analytes. Furthermore, these proteins are easy to produce, form pores with defined sizes, and can be easily manipulated with std. mol. biol. techniques. The range of possible analytes can be extended by using externally added adapter mols. Here, we provide an overview of current nanopore applications with a focus on engineering strategies and solns.
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  44. 44
    Diallinas, G. Understanding Transporter Specificity and the Discrete Appearance of Channel-like Gating Domains in Transporters. Front. Pharmacol. 2014,  DOI: 10.3389/fphar.2014.00207
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    Van De Cauter, L.; Fanalista, F.; Van Buren, L.; De Franceschi, N.; Godino, E.; Bouw, S.; Danelon, C.; Dekker, C.; Koenderink, G. H.; Ganzinger, K. A. Optimized CDICE for Efficient Reconstitution of Biological Systems in Giant Unilamellar Vesicles. ACS Synth. Biol. 2021, 10 (7), 1690– 1702,  DOI: 10.1021/acssynbio.1c00068
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    45
    Optimized cDICE for Efficient Reconstitution of Biological Systems in Giant Unilamellar Vesicles
    Van de Cauter, Lori; Fanalista, Federico; van Buren, Lennard; De Franceschi, Nicola; Godino, Elisa; Bouw, Sharon; Danelon, Christophe; Dekker, Cees; Koenderink, Gijsje H.; Ganzinger, Kristina A.
    ACS Synthetic Biology (2021), 10 (7), 1690-1702CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    Giant unilamellar vesicles (GUVs) are often used to mimic biol. membranes in reconstitution expts. They are also widely used in research on synthetic cells, as they provide a mech. responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomols. in GUVs, there is no one-size-fits-all soln. and reliable GUV fabrication still remains a major exptl. hurdle in the field. Here, we show that defect-free GUVs contg. complex biochem. systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of biol. systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (protein synthesis using recombinant elements) system. Our optimized cDICE method displays promising potential to become a std. method in biophysics and bottom-up synthetic biol.
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    Adamala, K. P.; Martin-Alarcon, D. A.; Guthrie-Honea, K. R.; Boyden, E. S. Engineering Genetic Circuit Interactions within and between Synthetic Minimal Cells. Nat. Chem. 2017, 9 (5), 431– 439,  DOI: 10.1038/nchem.2644
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    Engineering genetic circuit interactions within and between synthetic minimal cells
    Adamala, Katarzyna P.; Martin-Alarcon, Daniel A.; Guthrie-Honea, Katriona R.; Boyden, Edward S.
    Nature Chemistry (2017), 9 (5), 431-439CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
    Genetic circuits and reaction cascades are of great importance for synthetic biol., biochem. and bioengineering. An open question is how to maximize the modularity of their design to enable the integration of different reaction networks and to optimize their scalability and flexibility. One option is encapsulation within liposomes, which enables chem. reactions to proceed in well-isolated environments. Here we adapt liposome encapsulation to enable the modular, controlled compartmentalization of genetic circuits and cascades. We demonstrate that it is possible to engineer genetic circuit-contg. synthetic minimal cells (synells) to contain multiple-part genetic cascades, and that these cascades can be controlled by external signals as well as inter-liposomal communication without crosstalk. We also show that liposomes that contain different cascades can be fused in a controlled way so that the products of incompatible reactions can be brought together. Synells thus enable a more modular creation of synthetic biol. cascades, an essential step towards their ultimate programmability.
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  47. 47
    Rampioni, G.; D’Angelo, F.; Leoni, L.; Stano, P. Gene-Expressing Liposomes as Synthetic Cells for Molecular Communication Studies. Front. Bioeng. Biotechnol. 2019,  DOI: 10.3389/fbioe.2019.00001
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    Frallicciardi, J.; Melcr, J.; Siginou, P.; Marrink, S. J.; Poolman, B. Membrane Thickness, Lipid Phase and Sterol Type Are Determining Factors in the Permeability of Membranes to Small Solutes. Nat. Commun. 2022, 13 (1), 1– 12,  DOI: 10.1038/s41467-022-29272-x
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    Díaz-álvarez, A. E.; Cadierno, V. Glycerol: A Promising Green Solvent and Reducing Agent for Metal-Catalyzed Transfer Hydrogenation Reactions and Nanoparticles Formation. Appl. Sci. 2013, 3 (1), 55– 69,  DOI: 10.3390/app3010055
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    Glycerol: a promising green solvent and reducing agent for metal-catalyzed transfer hydrogenation reactions and nanoparticles formation
    Diaz-Alvarez, Alba E.; Cadierno, Victorio
    Applied Sciences (2013), 3 (1), 55-69CODEN: ASPCC7; ISSN:2076-3417. (MDPI AG)
    Glycerol is a non-toxic, non-hazardous, non-volatile, biodegradable, and recyclable liq. that is generated as a byproduct in the manuf. of biodiesel fuel from vegetable oils. Due to its easy availability, along with its unique combination of phys. and chem. properties, glycerol has recently emerged as an economically appealing and safe solvent for org. synthesis. Recent works have also demonstrated that glycerol can be used as a hydrogen source in metal-catalyzed transfer hydrogenation of org. compds., such as aldehydes, ketones, olefins and nitroarenes. Herein, the advances reached in this emerging field are reviewed. The utility of glycerol as solvent and reducing agent for the generation of metal nanoparticles is also briefly discussed.
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  50. 50
    Azua, A.; Finn, M.; Yi, H.; Beatriz Dantas, A.; Voutchkova-Kostal, A. Transfer Hydrogenation from Glycerol: Activity and Recyclability of Iridium and Ruthenium Sulfonate-Functionalized N-Heterocyclic Carbene Catalysts. ACS Sustain. Chem. Eng. 2017, 5 (5), 3963– 3972,  DOI: 10.1021/acssuschemeng.6b03156
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    Śmigiel, W. M.; Lefrançois, P.; Poolman, B. Physicochemical Considerations for Bottom-up Synthetic Biology. Emerg. Top. Life Sci. 2019, 3 (5), 445– 458,  DOI: 10.1042/ETLS20190017
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    Physicochemical considerations for bottom-up synthetic biology
    Smigiel, Wojciech Mikolaj; Lefrancois, Pauline; Poolman, Bert
    Emerging Topics in Life Sciences (2019), 3 (5), 445-458CODEN: ETLSAG; ISSN:2397-8562. (Portland Press Ltd.)
    A review. The bottom-up construction of synthetic cells from mol. components is arguably one of the most challenging areas of research in the life sciences. We review the impact of confining biol. systems in synthetic vesicles. Complex cell-like systems require control of the internal pH, ionic strength, (macro)mol. crowding, redox state and metabolic energy conservation. These physicochem. parameters influence protein activity and need to be maintained within limits to ensure the system remains in steady-state. We present the physicochem. considerations for building synthetic cells with dimensions ranging from the smallest prokaryotes to eukaryotic cells.
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  52. 52
    Maloney, P. C.; Kashket, E. R.; Wilson, T. H. A Protonmotive Force Drives ATP Synthesis in Bacteria. Proc. Natl. Acad. Sci. U. S. A. 1974, 71 (10), 3896– 3900,  DOI: 10.1073/pnas.71.10.3896
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    Protonmotive force drives ATP synthesis in bacteria
    Maloney, Peter C.; Kashket, E. R.; Wilson, T. Hastings
    Proceedings of the National Academy of Sciences of the United States of America (1974), 71 (10), 3896-900CODEN: PNASA6; ISSN:0027-8424.
    When cells of Streptococcus lactis or Escherichia coli were suspended in a K-free medium, a membrane potential (neg. inside) could be artificially generated by the addn. of the K ionophore, valinomycin. In response to this inward directed protonmotive force, ATP synthesis catalyzed by the membrane-bound ATPase (EC 3.6.1.3) was obsd. The formation of ATP was not found in S. lactis that had been treated with the ATPase inhibitor, N,N'-dicyclohexylcarbodiimide, nor was it obsd. in a mutant of E. coli lacking the ATPase. Inhibition of ATP synthesis by S. lactis was also obsd. when the membrane potential was reduced by the presence of external K, or when cells were first incubated with the proton conductor, carbonyl cyanide fluoromethoxyphenylhydrazone. These results are in agreement with predictions made by the chemiosmotic hypothesis of Mitchell.
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    Strahl, H.; Hamoen, L. W. Membrane Potential Is Important for Bacterial Cell Division. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (27), 12281– 12286,  DOI: 10.1073/pnas.1005485107
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    Membrane potential is important for bacterial cell division
    Strahl, Henrik; Hamoen, Leendert W.
    Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (27), 12281-12286, S12281/1-S12281/12CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Many cell division-related proteins are located at specific positions in the bacterial cell, and this organized distribution of proteins requires energy. Here, the authors report that the proton motive force, or more specifically the (trans)membrane potential, is directly involved in protein localization. It emerged that the membrane potential modulates the distribution of several conserved cell division proteins such as MinD, FtsA, and the bacterial cytoskeletal protein MreB. The authors show for MinD that this is based on the membrane potential stimulated binding of its C-terminal amphipathic helix. This function of the membrane potential has implications for how these morphogenetic proteins work and provide an explanation for the effects obsd. with certain antimicrobial compds.
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    Bruice, T. C. A View at the Millennium: The Efficiency of Enzymatic Catalysis. Acc. Chem. Res. 2002, 35 (3), 139– 148,  DOI: 10.1021/ar0001665
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    A view at the millennium: the efficiency of enzymatic catalysis
    Bruice, Thomas C.
    Accounts of Chemical Research (2002), 35 (3), 139-148CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
    A review and discussion with 58 refs. L. Pauling (1946) proposed that the active site of an enzyme (E) binds the transition state (TS) in preference to the substrate (S), and by doing so, stabilizes the TS and lowers the activation energy. E binding of TS in preference to S and increasing the TΔS⧧ by freezing out motions in E·S and E·TS have been accepted as the driving forces in enzymic catalysis. However, the smaller value of ΔG⧧ for a 1-substrate enzymic reaction, as compared to its nonenzymic counterpart, is generally the result of a smaller value of ΔH⧧. The TS in an enzymic reaction is reached through ground-state conformers that closely resemble the TS (near-attack conformers or NACs). E·NACs are in thermal equil. with all other E·S conformers and are turnstiles through which substrate mols. must pass to arrive at the lowest-energy TS. The TS in E·TS may or may not be bound tighter than the NAC is in E·NAC. Thus, the belief that enzymic reactions owe their facility to TS binding or to an increase in ΔStmo requires modification.
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    Feng, C.; Tollin, G.; Enemark, J. H. Sulfite Oxidizing Enzymes. Biochim. Biophys. Acta - Proteins Proteomics 2007, 1774 (5), 527– 539,  DOI: 10.1016/j.bbapap.2007.03.006
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    Sulfite oxidizing enzymes
    Feng, Changjian; Tollin, Gordon; Enemark, John H.
    Biochimica et Biophysica Acta, Proteins and Proteomics (2007), 1774 (5), 527-539CODEN: BBAPBW; ISSN:1570-9639. (Elsevier Ltd.)
    A review. Sulfite-oxidizing enzymes are essential mononuclear molybdoproteins involved in S metab. in animals, plants, and bacteria. There are 3 such enzymes presently known: (1) sulfite oxidase (SO) in animals, (2) SO in plants, and (3) sulfite dehydrogenase (SDH) in bacteria. X-ray crystal structures of enzymes from all 3 sources (chicken SO, Arabidopsis thaliana SO, and Starkeya novella SDH) show nearly identical square pyramidal coordination around the Mo atom, even though the overall structures of the proteins and the presence of addnl. cofactors vary. This structural information provides a mol. basis for studying the role of specific amino acids in catalysis. Animal SO catalyzes the final step in the degrdn. of S-contg. amino acids and is crit. in detoxifying excess sulfite. Human SO deficiency is a fatal genetic disorder that leads to early death, and impaired SO activity is implicated in sulfite neurotoxicity. Animal SO and bacterial SDH contain both Mo and heme domains, whereas plant SO only has the Mo domain. Intraprotein electron transfer (IET) between the Mo and Fe centers in animal SO and bacterial SDH is a key step in the catalysis, which can be studied by laser flash photolysis in the presence of deazariboflavin. IET studies on animal SO and bacterial SDH clearly demonstrate the similarities and differences between these 2 types of sulfite-oxidizing enzymes. Conformational change is involved in the IET of animal SO, in which electrostatic interactions may play a major role in guiding the docking of the heme domain to the Mo domain prior to electron transfer. In contrast, IET measurements for SDH demonstrate that IET occurs directly through the protein medium, which is distinctly different from that in animal SO. Point mutations in human SO can result in significantly impaired IET or no IET, thus rationalizing their fatal effects. The recent developments in the understanding of sulfite-oxidizing enzyme mechanisms that are driven by a combination of mol. biol., rapid kinetics, pulsed ESR, and computational techniques are the subject of this review.
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  56. 56
    Gong, W.; Hao, B.; Wei, Z.; Ferguson, D. J.; Tallant, T.; Krzycki, J. A.; Chan, M. K. Structure of the A2ε2 Ni-Dependent CO Dehydrogenase Component of the Methanosarcina Barkeri Acetyl-CoA Decarbonylase/Synthase Complex. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (28), 9558– 9563,  DOI: 10.1073/pnas.0800415105
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    56
    Structure of the α2ε2 Ni-dependent CO dehydrogenase component of the Methanosarcina barkeri acetyl-CoA decarbonylase/synthase complex
    Gong, Weimin; Hao, Bing; Wei, Zhiyi; Ferguson, Donald J., Jr.; Tallant, Thomas; Krzycki, Joseph A.; Chan, Michael K.
    Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (28), 9558-9563CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Ni-dependent carbon monoxide dehydrogenases (Ni-CODHs) are a diverse family of enzymes that catalyze reversible CO:CO2 oxidoreductase activity in acetogens, methanogens, and some CO-using bacteria. Crystallog. of Ni-CODHs from CO-using bacteria and acetogens has revealed the overall fold of the Ni-CODH core and has suggested structures for the C cluster that mediates CO:CO2 interconversion. Despite these advances, the mechanism of CO oxidn. has remained elusive. Herein, we report the structure of a distinct class of Ni-CODH from methanogenic archaea: the α2ε2 component from the α8β8γ8δ8ε8 CODH/acetyl-CoA decarbonylase/synthase complex, an enzyme responsible for the majority of biogenic methane prodn. on Earth. The structure of this Ni-CODH component provides support for a hitherto unobserved state in which both CO and H2O/OH- bind to the Ni and the exogenous FCII iron of the C cluster, resp., and offers insight into the structures and functional roles of the ε-subunit and FeS domain not present in nonmethanogenic Ni-CODHs.
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  57. 57
    Wilcoxen, J.; Zhang, B.; Hille, R. Reaction of the Molybdenum- and Copper-Containing Carbon Monoxide Dehydrogenase from Oligotropha Carboxydovorans with Quinones. Biochemistry 2011, 50 (11), 1910– 1916,  DOI: 10.1021/bi1017182
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    57
    Reaction of the Molybdenum- and Copper-Containing Carbon Monoxide Dehydrogenase from Oligotropha carboxydovorans with Quinones
    Wilcoxen, Jarett; Zhang, Bo; Hille, Russ
    Biochemistry (2011), 50 (11), 1910-1916CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Carbon monoxide dehydrogenase (CODH) from Oligotropha carboxydovorans catalyzes the oxidn. of carbon monoxide to carbon dioxide, providing the organism both a carbon source and energy for growth. In the oxidative half of the catalytic cycle, electrons gained from CO are ultimately passed to the electron transport chain of the Gram-neg. organism, but the proximal acceptor of reducing equiv. from the enzyme has not been established. Here we investigate the reaction of the reduced enzyme with various quinones and find them to be catalytically competent. Benzoquinone has a kox of 125.1 s-1 and a Kd of 48 μM. Ubiquinone-1 has a kox/Kd value of 2.88 × 105 M-1 s-1. 1,4-Naphthoquinone has a kox of 38 s-1 and a Kd of 140 μM, and 1,2-Naphthoquinone-4-sulfonic acid has a kox/Kd of 1.31 × 105 M-1 s-1. An extensive effort to identify a cytochrome that could be reduced by CO/CODH was unsuccessful. Steady-state studies with benzoquinone indicate that the rate-limiting step is in the reductive half of the reaction (i.e., the reaction of oxidized enzyme with CO). On the basis of the inhibition of CODH by diphenyliodonium chloride, we conclude that quinone substrates interact with CODH at the enzyme's flavin site. Our results strongly suggest that CODH donates reducing equiv. directly to the quinone pool without using a cytochrome as an intermediary.
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  58. 58
    Nishimura, H.; Nomura, Y.; Iwata, E.; Sato, N.; Sako, Y. Purification and Characterization of Carbon Monoxide Dehydrogenase from the Aerobic Hyperthermophilic Archaeon Aeropyrum Pernix. Fish. Sci. 2010, 76 (6), 999– 1006,  DOI: 10.1007/s12562-010-0277-8
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    Purification and characterization of carbon monoxide dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix
    Nishimura, Hiroshi; Nomura, Yoshiko; Iwata, Eri; Sato, Nozomi; Sako, Yoshihiko
    Fisheries Science (Tokyo, Japan) (2010), 76 (6), 999-1006CODEN: FSCIEH; ISSN:0919-9268. (Springer Japan)
    The aerobic hyperthermophilic archaeon Aeropyrum pernix expresses carbon monoxide (CO) oxidn. activity under heterotrophic growth conditions. Using activity stain gel anal., CO oxidn. activity was detected in a protein with a mol. mass of 210 kDa. The 210 kDa CODH protein was purified to homogeneity from A. pernix. Aeropyrum Mo-CODH catalyzed the oxidn. of CO with a specific activity of 2.1 μmol CO min-1 mg-1 at 95°C, pH 8.0 using Me viologen as the electron acceptor. The CODH protein showed high oxygen and thermo stability. The protein contains three subunits: L (86.6 kDa), M (34.5 kDa), and S (12.6 kDa), which form the LM2S complex. The mol. mass of the complex was calcd. by gel filtration and found to be 163.7 kDa. N-terminal amino acid sequencing and peptide mass fingerprinting anal. of the subunits indicated that they corresponded to NP_148462.1, NP_148464.2, and NP_148465.1, and their genes annotated the molybdo iron-sulfur flavoprotein carbon monoxide dehydrogenase S, L, and M subunits, resp. Phylogenetic anal. revealed that CODH belongs to a novel clade of diverse CODHs.
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    Ruzheinikov, S. N.; Burke, J.; Sedelnikova, S.; Baker, P. J.; Taylor, R.; Bullough, P. A.; Muir, N. M.; Gore, M. G.; Rice, D. W. Glycerol Dehydrogenase: Structure, Specificity, and Mechanism of a Family III Polyol Dehydrogenase. Structure 2001, 9 (9), 789– 802,  DOI: 10.1016/S0969-2126(01)00645-1
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    Glycerol dehydrogenase structure, specificity, and mechanism of a family III polyol dehydrogenase
    Ruzheinikov, S. N.; Burke, J.; Sedelnikova, S.; Baker, P. J.; Taylor, R.; Bullough, P. A.; Muir, N. M.; Gore, M. G.; Rice, D. W.
    Structure (Cambridge, MA, United States) (2001), 9 (9), 789-802CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
    Bacillus stearothermophilus glycerol dehydrogenase (EC 1.1.1.6) (I) catalyzes the oxidn. of glycerol to dihydroxyacetone with the concomitant redn. of NAD to NADH. Anal. of the sequence of this enzyme indicates that it is a member of the so-called Fe-contg. alc. dehydrogenase family. Despite this sequence similarity, I shows a strict dependence on Zn for activity. On the basis of this, the authors propose to rename this group the family III metal-dependent polyol dehydrogenases. To date, no structural data have been reported for any enzyme in this group. Here, the crystal structure of B. stearothermophilus I was detd. at 1.7 Å resoln. to provide structural insights into the mechanistic features of this family. I has 370 amino acid residues, has a mol. wt. of 39.5 kDa, and is a homooctamer in soln. Anal. of the crystal structures of the free enzyme and of its binary complexes with NAD and glycerol showed that the active site of I was in the cleft between the enzyme's 2 domains, with the catalytic Zn2+ ion playing a role in stabilizing an alkoxide intermediate. In addn., the specificity of I for a range of diols could be understood, as both OH groups of glycerol formed ligands to the enzyme-bound Zn2+ ion at the active site. The structure further revealed a previously unsuspected similarity to dehydroquinate synthase, an enzyme whose more complex chem. shares a common chem. step with that catalyzed by I, providing a striking example of divergent evolution. Finally, the structure suggested that the NAD-binding domain of I may be related to that of the classical Rossmann fold by switching the sequence order of the 2 mononucleotide binding folds that make up this domain.
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    Heck, d. H. A.; Casanova, M.; Starr, T. B. Formaldehyde Toxicity - New Understanding. Crit. Rev. Toxicol. 1990, 20 (6), 397– 426,  DOI: 10.3109/10408449009029329
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    Formaldehyde toxicity. New understanding
    Heck, Henry d'A.; Casanova, Mercedes; Starr, Thomas B.
    Critical Reviews in Toxicology (1990), 20 (6), 397-426CODEN: CRTXB2; ISSN:0045-6446.
    A review with 235 refs. on metab., toxicity, mutagenicity, reactions with macromols., mol. dosimetry, epidemiol., and risk assessment of formaldehyde.
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    Muñoz-Elías, E. J.; McKinney, J. D. Carbon Metabolism of Intracellular Bacteria. Cell. Microbiol. 2006, 8 (1), 10– 22,  DOI: 10.1111/j.1462-5822.2005.00648.x
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    Carbon metabolism of intracellular bacteria
    Munoz-Elias, Ernesto J.; McKinney, John D.
    Cellular Microbiology (2006), 8 (1), 10-22CODEN: CEMIF5; ISSN:1462-5814. (Blackwell Publishing Ltd.)
    A review. Bacterial metab. was studied intensively since the 1st observations of these 'animalcules' by Leeuwenhoek and their isolation in pure cultures by Pasteur. Metabolic studies have traditionally focused on a small no. of model organisms, primarily the Gram neg. bacillus Escherichia coli, adapted to artificial culture conditions in the lab. Comparatively little is known about the physiol. and metab. of wild microorganisms living in their natural habitats. For ∼500-1000 species of commensals and symbionts, and a smaller no. of pathogenic bacteria, that habitat is the human body. Emerging evidence suggests that the metab. of bacteria grown in vivo differs profoundly from their metab. in axenic cultures.
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    Hochstein, L. I.; Tomlinson, G. A. The Enzymes Associated with Denitrification. Annu. Rev. Microbiol. 1988, 42 (72), 231– 261,  DOI: 10.1146/annurev.mi.42.100188.001311
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    The enzymes associated with denitrification
    Hochstein, Lawrence I.; Tomlinson, Geraldine A.
    Annual Review of Microbiology (1988), 42 (), 231-61CODEN: ARMIAZ; ISSN:0066-4227.
    A review with 170 refs. on the enzyme systems which confer upon some eubacteria and archaebacteria the ability to grow anaerobically by reducing ionic nitrogenous oxides to gaseous products. The topics discussed include nitrate reductase, nitrite reductases, nitric oxide reductases, mechanism of N-N bond formation, and nitrous oxide reductases.
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    Dolla, A.; Fournier, M.; Dermoun, Z. Oxygen Defense in Sulfate-Reducing Bacteria. J. Biotechnol. 2006, 126 (1), 87– 100,  DOI: 10.1016/j.jbiotec.2006.03.041
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    Oxygen defense in sulfate-reducing bacteria
    Dolla, Alain; Fournier, Marjorie; Dermoun, Zorah
    Journal of Biotechnology (2006), 126 (1), 87-100CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)
    A review. Sulfate-reducing bacteria (SRB) are strict anaerobes that are often found in biotopes where oxic conditions can temporarily exist. The bacteria have developed several defense strategies to survive exposure to oxygen. These strategies includes peculiar behaviors in the presence of oxygen, like aggregation or aerotaxis, and enzymic systems dedicated to the redn. and the elimination of oxygen and its reactive species. Sulfate-reducing bacteria, and specially Desulfovibrio species, possess a variety of enzymes acting together to achieve an efficient defense against oxidative stress. The function and occurrence of these enzymic systems are described.
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    Lu, Z.; Imlay, J. A. When Anaerobes Encounter Oxygen: Mechanisms of Oxygen Toxicity, Tolerance and Defence. Nat. Rev. Microbiol. 2021, 19 (12), 774– 785,  DOI: 10.1038/s41579-021-00583-y
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    When anaerobes encounter oxygen: mechanisms of oxygen toxicity, tolerance and defence
    Lu, Zheng; Imlay, James A.
    Nature Reviews Microbiology (2021), 19 (12), 774-785CODEN: NRMACK; ISSN:1740-1526. (Nature Portfolio)
    Abstr.: The defining trait of obligate anaerobes is that oxygen blocks their growth, yet the underlying mechanisms are unclear. A popular hypothesis was that these microorganisms failed to evolve defences to protect themselves from reactive oxygen species (ROS) such as superoxide and hydrogen peroxide, and that this failure is what prevents their expansion to oxic habitats. However, studies reveal that anaerobes actually wield most of the same defences that aerobes possess, and many of them have the capacity to tolerate substantial levels of oxygen. Therefore, to understand the structures and real-world dynamics of microbial communities, investigators have examd. how anaerobes such as Bacteroides, Desulfovibrio, Pyrococcus and Clostridium spp. struggle and cope with oxygen. The hypoxic environments in which these organisms dwell - including the mammalian gut, sulfur vents and deep sediments - experience episodic oxygenation. In this Review, we explore the mol. mechanisms by which oxygen impairs anaerobes and the degree to which bacteria protect their metabolic pathways from it. The emergent view of anaerobiosis is that optimal strategies of anaerobic metab. depend upon radical chem. and low-potential metal centers. Such catalytic sites are intrinsically vulnerable to direct poisoning by mol. oxygen and ROS. Observations suggest that anaerobes have evolved tactics that either minimize the extent to which oxygen disrupts their metab. or restore function shortly after the stress has dissipated.
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    Patil, Y. P.; Jadhav, S. Novel Methods for Liposome Preparation. Chem. Phys. Lipids 2014, 177, 8– 18,  DOI: 10.1016/j.chemphyslip.2013.10.011
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    Novel methods for liposome preparation
    Patil, Yogita P.; Jadhav, Sameer
    Chemistry and Physics of Lipids (2014), 177 (), 8-18CODEN: CPLIA4; ISSN:0009-3084. (Elsevier Ltd.)
    A review. Liposomes are bilayer vesicles which have found use, among other applications, as drug delivery vehicles. Conventional techniques for liposome prepn. and size redn. remain popular as these are simple to implement and do not require sophisticated equipment. However, issues related to scale-up for industrial prodn. and scale-down for point-of-care applications have motivated improvements to conventional processes and have also led to the development of novel routes to liposome formation. In this article, these modified and new methods for liposome prepn. have been reviewed and classified with the objective of updating the reader to recent developments in liposome prodn. technol.
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    Mitchell, P. Coupling of Phosphorylation to Electron and Hydrogen Transfer by a Chemi-Osmotic Type of Mechanism. Nature 1961, 191 (4784), 144– 148,  DOI: 10.1038/191144a0
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    Coupling of phosphorylation to electron and hydrogen transfer by a chemiosmotic type of mechanism
    Mitchell, Peter
    Nature (London, United Kingdom) (1961), 191 (), 144-8CODEN: NATUAS; ISSN:0028-0836.
    A chemiosmotic mechanism dependent on the supramol. organization (membrane structure) of multienzyme systems is proposed in contrast to the orthodox substrate-enzyme type of coupling. The driving force is postulated as due to spatially directed diffusion of the active components. An essential requirement is the presence within an ion-impermeable membrane of an anisotropic reversible adenosine triphosphatase (ATPase) system (active center). In such an active center (e.g. phosphokinase), the hydrolysis equil. in the adenosine triphosphate (ATP)-adenosine diphosphate (ADP) system is detd. by the electrochem. activity of the H2O at the active center, [H2O]c = [H+]R[OH]L = [H+]R[H2O]aq./[H+]L where R (right) and L (left) designate the cytoplasmic and intracellular aq. phases, resp. The electrochem. activity ratio for the enzyme system, including the elements of H2O, is given as: [ATP]/[ADP] = {[P]/K[H2O]} {[H+]L/[H+]R}. At pH 7, K1[H2O]aq. ∼105; where [P] is at physiol. levels (approx. 10-2M), the ratio is: [ATP]/[ADP] equal or nearly equal to {[H+]L/[H+]R} × 10-7. Reversal of enzyme activity is kinetically explained on the basis of the electrochem. activity gradient of H+ and OH- across the active center. The fundamental processes essentially involve dehydroxylation and deprotonation. The [H+] and [OH-] gradients are governed by an anisotropic electron-chain transfer (reoxidn.-redn.) mechanism coupled to the reversible ATPase system of the active center. One ATP mol. is produced per electron transfer when the oxidn.-redn. and phosphorylation systems are in chemiosmotic equil. The energy relation is expressed as [ATP]/[ADP] = {[P]/105} × 10ΔE/60, where ΔE (the oxidn.-redn. span) is equiv. to the free energy change in mv./electron transferred. At inorg. phosphate concns. of 10-2M this would require a min. ΔE of 420 mv. to drive the ATP synthesis. Such energies are readily available through the coenzyme (nucleotides, flavoproteins, ubiquinones) and the carboxylic acid (succinic-fumarate) systems. Diagrammatic representations are shown for the coupling systems. Loose membrane structures (or equiv. uncoupling agents like dinitrophenols) are postulated as disrupting the chemiosmotic coupling mechanism with consequent change in the energy relations of the steady state. The chemiosmotic hypothesis is utilized to explain photophosphorylation and also a number of other facts (absence of energy-rich intermediates; dependence of coupling on membrane structure and its ion impermeability; differential effects of [H+]; action of uncoupling agents; membrane swelling and shrinkage) difficult to reconcile on the basis of classical concepts. In some concluding speculations, membrane transport and metabolism are considered as simply different aspects of a unifying process, termed vectorial metabolism.
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    Russell, J. B. The Energy Spilling Reactions of Bacteria and Other Organisms. J. Mol. Microbiol. Biotechnol. 2007, 13 (1–3), 1– 11,  DOI: 10.1159/000103591
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    The energy spilling reactions of bacteria and other organisms
    Russell, James B.
    Journal of Molecular Microbiology and Biotechnology (2007), 13 (1-3), 1-11CODEN: JMMBFF; ISSN:1464-1801. (S. Karger AG)
    A review. For many years, it was assumed that living organisms always utilized ATP in a highly efficient manner, but simple growth studies with bacteria indicated that the efficiency of biomass prodn. was often at least 3-fold lower than the amt. that would be predicted from std. biosynthetic pathways. The utilization of energy for maintenance could only explain a small portion of this discrepancy particularly when the growth rate was high. These ideas and thermodn. arguments indicated that cells might have another avenue of energy utilization. This phenomenon has also been called 'uncoupling', 'spillage' and 'overflow metab.', but 'energy spilling' is probably the most descriptive term. It appears that many bacteria spill energy, and the few that do not can be killed (large and often rapid decrease in viability), if the growth medium is nitrogen-limited and the energy source is in 'excess'. The lactic acid bacterium, Streptococcus bovis, is an ideal bacterium for the study of energy spilling. Because it only uses substrate level phosphorylation to generate ATP, ATP generation can be calcd. with a high degree of certainty. It does not store glucose as glycogen, and its cell membrane can be easily accessed. Comparative anal. of heat prodn., membrane voltage, ATP prodn. and Ohm's law indicated that the energy spilling reaction of S. bovis is mediated by a futile cycle of protons through the cell membrane. Less is known about Escherichia coli, but in this bacterium energy spilling could be mediated by a futile cycle of potassium or ammonium ions. Energy spilling is not restricted to prokaryotes and appears to occur in yeasts and in higher organisms. In man, energy spilling may be related to cancer, aging, ischemia and cardiac failure.
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    Jin, Q. Energy Conservation of Anaerobic Respiration. Am. J. Sci. 2012, 312 (6), 573– 628,  DOI: 10.2475/06.2012.01
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    Energy conservation of anaerobic respiration
    Jin, Qusheng
    American Journal of Science (2012), 312 (6), 573-628CODEN: AJSCAP; ISSN:0002-9599. (Kline Geology Laboratory)
    A review. Microbes save energy from the environment by synthesizing ATP. Understanding microbial energy conservation is important for both theor. and practical applications. This paper focuses on the common metab. of anoxic environments-ferric iron respiration, sulfate respiration, and methanogenesis-and analyzes microbial energy conservation on the basis of the thermodn. as well as physiol. models of respiratory reactions. The results of the anal. show that iron respiration synthesizes 1 to 4 ATPs by transferring eight electrons from H2, acetate, lactate, and ethanol to ferric minerals; sulfate respiration makes 0.25 to more than 3 ATPs by transferring eight electrons from the same suite of electron donors to sulfate; methanogenesis yields 0 to 1 ATP by oxidizing four H2 and by disproportionating one acetate. The ATP yields are compared to growth yields and energy thresholds of anaerobic metab. to explore the impact of energy conservation. Specifically, energy conservation controls microbial growth: in geochem. systems, respiring microbes synthesize up to 5 g biomass per mol of ATP. Energy conservation also requires the environment to supply chem. energy at quantities greater than the energy saved by microbes. These results unify our view of microbial metab., and can be applied to evaluating the occurrence and significance of microbial life in natural environments.
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    Jones, A. J. Y.; Blaza, J. N.; Bridges, H. R.; May, B.; Moore, A. L.; Hirst, J. A Self-Assembled Respiratory Chain That Catalyzes NADH Oxidation by Ubiquinone-10 Cycling between ComplexI and the Alternative Oxidase. Angew. Chemie - Int. Ed. 2016, 55 (2), 728– 731,  DOI: 10.1002/anie.201507332
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    A Self-Assembled Respiratory Chain that Catalyzes NADH Oxidation by Ubiquinone-10 Cycling between Complex I and the Alternative Oxidase
    Jones, Andrew J. Y.; Blaza, James N.; Bridges, Hannah R.; May, Benjamin; Moore, Anthony L.; Hirst, Judy
    Angewandte Chemie, International Edition (2016), 55 (2), 728-731CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Complex I is a crucial respiratory enzyme that conserves the energy from NADH oxidn. by ubiquinone-10 (Q10) in proton transport across a membrane. Studies of its energy transduction mechanism are hindered by the extreme hydrophobicity of Q10, and they have so far relied on native membranes with many components or on hydrophilic Q10 analogs that partition into membranes and undergo side reactions. Herein, we present a self-assembled system without these limitations: proteoliposomes contg. mammalian complex I, Q10, and a quinol oxidase (the alternative oxidase, AOX) to recycle Q10H2 to Q10. AOX is present in excess, so complex I is completely rate detg. and the Q10 pool is kept oxidized under steady-state catalysis. The system was used to measure a fully-defined KM value for Q10. The strategy is suitable for any enzyme with a hydrophobic quinone/quinol substrate, and could be used to characterize hydrophobic inhibitors with potential applications as pharmaceuticals, pesticides, or fungicides.
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    Biner, O.; Fedor, J. G.; Yin, Z.; Hirst, J. Bottom-Up Construction of a Minimal System for Cellular Respiration and Energy Regeneration. ACS Synth. Biol. 2020, 9 (6), 1450– 1459,  DOI: 10.1021/acssynbio.0c00110
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    Bottom-Up Construction of a Minimal System for Cellular Respiration and Energy Regeneration
    Biner, Olivier; Fedor, Justin G.; Yin, Zhan; Hirst, Judy
    ACS Synthetic Biology (2020), 9 (6), 1450-1459CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    ATP, the cellular energy currency, is essential for life. The ability to provide a const. supply of ATP is therefore crucial for the construction of artificial cells in synthetic biol. Here, the authors describe the bottom-up assembly and characterization of a minimal respiratory system that uses NADH as a fuel to produce ATP from ADP and inorg. phosphate, and is thus capable of sustaining both upstream metabolic processes that rely on NAD+, and downstream energy-demanding processes that are powered by ATP hydrolysis. A detergent-mediated approach was used to coreconstitute respiratory mitochondrial complex I and an F-type ATP synthase into nanosized liposomes. Addn. of the alternative oxidase to the resulting proteoliposomes produced a minimal artificial "organelle" that reproduces the energy-converting catalytic reactions of the mitochondrial respiratory chain: NADH oxidn., ubiquinone cycling, oxygen redn., proton pumping, and ATP synthesis. As a proof-of-principle, the authors demonstrate that the nanovesicles are capable of using an NAD+-linked substrate to drive cell-free protein expression. The nanovesicles are both efficient and durable and may be applied to sustain artificial cells in future work.
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    Norris, F. A.; Powell, G. L. The Apparent Permeability Coefficient for Proton Flux through Phosphatidylcholine Vesicles Is Dependent on the Direction of Flux. BBA - Biomembr. 1990, 1030 (1), 165– 171,  DOI: 10.1016/0005-2736(90)90252-J
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    Garcia Costas, A. M.; White, A. K.; Metcalf, W. W. Purification and Characterization of a Novel Phosphorus-Oxidizing Enzyme from Pseudomonas Stutzeri WM88. J. Biol. Chem. 2001, 276 (20), 17429– 17436,  DOI: 10.1074/jbc.M011764200
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    Purification and characterization of a novel phosphorus-oxidizing enzyme from Pseudomonas stutzeri WM88
    Garcia Costas, Amaya M.; White, Andrea K.; Metcalf, William W.
    Journal of Biological Chemistry (2001), 276 (20), 17429-17436CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The ptxD gene from Pseudomonas stutzeri WM88 encoding the novel phosphorus oxidizing enzyme NAD:phosphite oxidoreductase (trivial name phosphite dehydrogenase, PtxD) was cloned into an expression vector and over-produced in Escherichia coli. The heterologously produced enzyme is indistinguishable from the native enzyme based on mass spectrometry, amino-terminal sequencing, and specific activity analyses. Recombinant PtxD was purified to homogeneity via a two-step affinity protocol and characterized. The enzyme stoichiometrically produces NADH and phosphate from NAD and phosphite. The reverse reaction was not obsd. Gel filtration anal. of the purified protein is consistent with PtxD acting as a homodimer. PtxD has a high affinity for its substrates with Km values of 53.1 ± 6.7 μM and 54.6 ± 6.7 μM, for phosphite and NAD, resp. Vmax and kcat were detd. to be 12.2 ± 0.3 μmol min-1 mg-1 and 440 min-1. NADP can substitute poorly for NAD; however, none of the numerous compds. examd. were able to substitute for phosphite. Initial rate studies in the absence or presence of products and in the presence of the dead end inhibitor sulfite are most consistent with a sequential ordered mechanism for the PtxD reaction, with NAD binding first and NADH being released last. Amino acid sequence comparisons place PtxD as a new member of the D-2-hydroxyacid NAD-dependent dehydrogenases, the only one to have an inorg. substrate. To our knowledge, this is the first detailed biochem. study on an enzyme capable of direct oxidn. of a reduced phosphorus compd.
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    NAD+-dependent formate dehydrogenase
    Popov, Vladimir O.; Lamzin, Victor S.
    Biochemical Journal (1994), 301 (3), 625-43CODEN: BIJOAK; ISSN:0264-6021.
    A review with 158 refs., of the kinetic properties and structure of the title enzyme.
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  74. 74
    Tishkov, V. I.; Popov, V. O. Catalytic Mechanism and Application of Formate Dehydrogenase. Biochem. 2004, 69 (11), 1252– 1267,  DOI: 10.1007/s10541-005-0071-x
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    Catalytic mechanism and application of formate dehydrogenase
    Tishkov, V. I.; Popov, V. O.
    Biochemistry (Moscow, Russian Federation) (2004), 69 (11), 1252-1267, 1 plateCODEN: BIORAK; ISSN:0006-2979. (MAIK Nauka/Interperiodica Publishing)
    A review. NAD-dependent formate dehydrogenase (FDH) is an abundant enzyme that plays an important role in energy supply of methylotrophic microorganisms and in response to stress in plants. FDH belongs to the superfamily of D-specific 2-hydroxy acid dehydrogenases. FDH is widely accepted as a model enzyme to study the mechanism of hydride ion transfer in the active center of dehydrogenases because the reaction catalyzed by the enzyme is devoid of proton transfer steps and implies a substrate with relatively simple structure. FDH is also widely used in enzymic syntheses of optically active compds. as a versatile biocatalyst for NAD(P)H regeneration consumed in the main reaction. This review covers recent developments in cloning genes of FDH from various sources, studies of its catalytic mechanism and physiol. role, and its application for new chiral syntheses.
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  75. 75
    Schneider, K.; Schlegel, H. G. Purification and Properties of Soluble Hydrogenase from Alcaligenes Eutrophus H 16. Biochim. Biophys. Acta (BBA)-Enzymology 1976, 452 (1), 66– 80,  DOI: 10.1016/0005-2744(76)90058-9
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    Burgdorf, T.; van der Linden, E.; Bernhard, M.; Yin, Q. Y.; Back, J. W.; Hartog, A. F.; Muijsers, A. O.; de Koster, C. G.; Albracht, S. P. J.; Friedrich, B. The Soluble NAD+-Reducing [NiFe]-Hydrogenase from Ralstonia Eutropha H16 Consists of Six Subunits and Can Be Specifically Activated by NADPH. J. Bacteriol. 2005, 187 (9), 3122– 3132,  DOI: 10.1128/JB.187.9.3122-3132.2005
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    76
    The soluble NAD+-reducing [NiFe]-hydrogenase from Ralstonia eutropha H16 consists of six subunits and can be specifically activated by NADPH
    Burgdorf, Tanja; van der Linden, Eddy; Bernhard, Michael; Yin, Qing Yuan; Back, Jaap W.; Hartog, Aloysius F.; Muijsers, Anton O.; de Koster, Chris G.; Albracht, Simon P. J.; Friedrich, Baerbel
    Journal of Bacteriology (2005), 187 (9), 3122-3132CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
    The sol. [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates redn. of NAD+ at the expense of H2. We have purified a new high-mol.-wt. form of the SH which contains an addnl. subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degrdn., in combination with protein sequencing of the SH high-mol.-wt. complex, established a subunit stoichiometry of HoxFUYHI2. Crosslinking expts. indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amts. of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amts. of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex I).
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  77. 77
    Herr, N.; Ratzka, J.; Lauterbach, L.; Lenz, O.; Ansorge-Schumacher, M. B. Stability Enhancement of an O2-Tolerant NAD+-Reducing [NiFe]-Hydrogenase by a Combination of Immobilisation and Chemical Modification. J. Mol. Catal. B Enzym. 2013, 97, 169– 174,  DOI: 10.1016/j.molcatb.2013.06.009
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    77
    Stability enhancement of an O2-tolerant NAD+-reducing [NiFe|-hydrogenase by a combination of immobilization and chemical modification
    Herr, Nicole; Ratzka, Juliane; Lauterbach, Lars; Lenz, Oliver; Ansorge-Schumacher, Marion B.
    Journal of Molecular Catalysis B: Enzymatic (2013), 97 (), 169-174CODEN: JMCEF8; ISSN:1381-1177. (Elsevier B.V.)
    The oxygen-tolerant, NAD+-reducing sol. hydrogenase (SH) from Ralstonia eutropha H16 is a promising catalyst for cofactor regeneration in enzyme-catalyzed redn. processes. The tech. use of the isolated enzyme, however, is limited by its relatively low stability under operational conditions such as agitation, elevated temp. or addn. of co-solvents. The max. half-life at a reaction temp. of 35° and pH 8.0 was only 5.3 h. In order to enhance the stability of the enzyme, it was immobilized onto the anionic resin Amberlite FPA54. At an immobilization yield of 93.4% for adsorptive and 100% for covalent attachment, corresponding activities of 48.9 and 39.3%, resp., were obtained. Covalent binding always yielded superior stabilization. At elevated temp. and under agitation, stabilization was further increased by modification of the covalently bound SH with methoxy-poly(ethylene) glycol (mPEG). A comparable effect was not achieved when SH modification was performed before immobilization. In stationary aq. soln., half-lives of up to 161 h at 25° and 32 h at 35° were obtained. In presence of the tech. relevant co-solvents DMSO, DMF, 2-propanol and [EMIM][EtSO4] half-lives of 14-29 h can now be achieved.
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  78. 78
    Velasco-Lozano, S.; Roca, M.; Leal-Duaso, A.; Mayoral, J. A.; Pires, E.; Moliner, V.; López-Gallego, F. Selective Oxidation of Alkyl and Aryl Glyceryl Monoethers Catalysed by an Engineered and Immobilised Glycerol Dehydrogenase. Chem. Sci. 2020, 11 (44), 12009– 12020,  DOI: 10.1039/D0SC04471G
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    78
    Selective oxidation of alkyl and aryl glyceryl monoethers catalysed by an engineered and immobilized glycerol dehydrogenase
    Velasco-Lozano, Susana; Roca, Maite; Leal-Duaso, Alejandro; Mayoral, Jose A.; Pires, Elisabet; Moliner, Vicent; Lopez-Gallego, Fernando
    Chemical Science (2020), 11 (44), 12009-12020CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    Enzymes acting over glyceryl ethers are scarce in living cells, and consequently biocatalytic transformations of these mols. are rare despite their interest for industrial chem. In this work, we have engineered and immobilized a glycerol dehydrogenase from Bacillus stearothermophilus (BsGlyDH) to accept a battery of alkyl/aryl glyceryl monoethers and catalyze their enantioselective oxidn. to yield the corresponding 3-alkoxy/aryloxy-1-hydroxyacetones. QM/MM computational studies decipher the key role of D123 in the oxidn. catalytic mechanism, and reveal that this enzyme is highly enantioselective towards S-isomers (ee > 99%). Through structure-guided site-selective mutagenesis, we find that the mutation L252A sculpts the active site to accommodate a productive configuration of 3-monoalkyl glycerols. This mutation enhances the kcat 163-fold towards 3-ethoxypropan-1,2-diol, resulting in a specific activity similar to the one found for the wild-type towards glycerol. Furthermore, we immobilized the L252A variant to intensify the process, demonstrating the reusability and increasing the operational stability of the resulting heterogeneous biocatalyst. Finally, we manage to integrate this immobilized enzyme into a one-pot chemoenzymic process to convert glycidol and ethanol into 3-ethoxy-1-hydroxyacetone and (R)-3-ethoxypropan-1,2-diol, without affecting the oxidn. activity. These results thus expand the uses of engineered glycerol dehydrogenases in applied biocatalysis for the kinetic resoln. of glycerol ethers and the manufg. of substituted hydroxyacetones.
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  79. 79
    Matelska, D.; Shabalin, I. G.; Jabłońska, J.; Domagalski, M. J.; Kutner, J.; Ginalski, K.; Minor, W. Classification, Substrate Specificity and Structural Features of D-2-Hydroxyacid Dehydrogenases: 2HADH Knowledgebase. BMC Evol. Biol. 2018, 18 (1), 1– 23,  DOI: 10.1186/s12862-018-1309-8
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    79
    Classification, substrate specificity and structural features of D-2-hydroxyacid dehydrogenases: 2HADH knowledgebase
    Matelska, Dorota; Shabalin, Ivan G.; Jablonska, Jagoda; Domagalski, Marcin J.; Kutner, Jan; Ginalski, Krzysztof; Minor, Wladek
    BMC Evolutionary Biology (2018), 18 (1), 1-23CODEN: BEBMCG; ISSN:1471-2148. (BioMed Central Ltd.)
    We report an in-depth phylogenetic anal., followed by mapping of available biochem. and structural data on the reconstructed phylogenetic tree. The anal. suggests that some subfamilies comprising enzymes with similar yet broad substrate specificity profiles diverged early in the evolution of 2HADHs. Based on the phylogenetic tree, we present a revised classification of the family that comprises 22 subfamilies, including 13 new subfamilies not studied biochem. We summarize characteristics of the nine biochem. studied subfamilies by aggregating all available sequence, biochem., and structural data, providing comprehensive descriptions of the active site, cofactor-binding residues, and potential roles of specific structural regions in substrate recognition. In addn., we concisely present our anal. as an online 2HADH enzymes knowledgebase. Conclusions: The knowledgebase enables navigation over the 2HADHs classification, search through collected data, and functional predictions of uncharacterized 2HADHs. Future characterization of the new subfamilies may result in discoveries of enzymes with novel metabolic roles and with properties beneficial for biotechnol. applications.
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  80. 80
    Woodyer, R.; Van der Donk, W. A.; Zhao, H. Relaxing the Nicotinamide Cofactor Specificity of Phosphite Dehydrogenase by Rational Design. Biochemistry 2003, 42 (40), 11604– 11614,  DOI: 10.1021/bi035018b
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    80
    Relaxing the nicotinamide cofactor specificity of phosphite dehydrogenase by rational design
    Woodyer, Ryan; van der Donk, Wilfred A.; Zhao, Huimin
    Biochemistry (2003), 42 (40), 11604-11614CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Homol. modeling was used to identify two particular residues, Glu175 and Ala176, in Pseudomonas stutzeri phosphite dehydrogenase(PTDH) as the principal determinants of nicotinamide cofactor (NAD+ and NADP+) specificity. Replacement of these two residues by site-directed mutagenesis with Ala175 and Arg176 both sep. and in combination resulted in PTDH mutants with relaxed cofactor specificity. All three mutants exhibited significantly better catalytic efficiency for both cofactors, with the best kinetic parameters displayed by the double mutant, which had a 3.6-fold higher catalytic efficiency for NAD+ and a 1000-fold higher efficiency for NADP+. The cofactor specificity was changed from 100-fold in favor of NAD+ for the wild-type enzyme to 3-fold in favor of NADP+ for the double mutant. Isoelec. focusing of the proteins in a nondenaturing gel showed that the replacement with more basic residues indeed changed the effective pI of the protein. HPLC anal. of the enzymic products of the double mutant verified that the reaction proceeded to completion using either substrate and produced only the corresponding reduced cofactor and phosphate. Thermal inactivation studies showed that the double mutant was protected from thermal inactivation by both cofactors, while the wild-type enzyme was protected by only NAD+. The combined results provide clear evidence that Glu175 and Ala176 are both crit. for nicotinamide cofactor specificity. The rationally designed double mutant might be useful for the development of an efficient in vitro NAD(P)H regeneration system for reductive biocatalysis.
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  81. 81
    Metcalf, W. W.; Wolfe, R. S. Molecular Genetic Analysis of Phosphite and Hypophosphite Oxidation by Pseudomonas Stutzeri WM88. J. Bacteriol. 1998, 180 (21), 5547– 5558,  DOI: 10.1128/JB.180.21.5547-5558.1998
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    81
    Molecular genetic analysis of phosphite and hypophosphite oxidation by Pseudomonas stutzeri WM88
    Metcalf, William W.; Wolfe, Ralph S.
    Journal of Bacteriology (1998), 180 (21), 5547-5558CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
    The first mol. and genetic characterization of a biochem. pathway for oxidn. of the reduced phosphorus (P) compds. phosphite and hypophosphite is reported. The pathway was identified in Pseudomonas stutzeri WM88, which was chosen for detailed studies from a group of organisms isolated based on their ability to oxidize hypophosphite (+1 valence) and phosphite (+3 valence) to phosphate (+5 valence). The genes required for oxidn. of both compds. by P. stutzeri WM88 were cloned on a single ca. 30-kbp DNA fragment by screening for expression in Escherichia coli and Pseudomonas aeruginosa. Two lines of evidence suggest that hypophosphite is oxidized to phosphate via a phosphite intermediate. First, plasmid subclones that conferred oxidn. of phosphite, but not hypophosphite, upon heterologous hosts were readily obtained. All plasmid subclones that failed to confer phosphite oxidn. also failed to confer hypophosphite oxidn. No subclones that conferred only hypophosphite expression were obtained. Second, various deletion derivs. of the cloned genes were made in vitro and recombined onto the chromosome of P. stutzeri WM88. Two phenotypes were displayed by individual mutants. Mutants with the region encoding phosphite oxidn. deleted (based upon the subcloning results) lost the ability to oxidize either phosphite or hypophosphite. Mutants with the region encoding hypophosphite oxidn. deleted lost only the ability to oxidize hypophosphite. The phenotypes displayed by these mutants also demonstrate that the cloned genes are responsible for the P oxidn. phenotypes displayed by the original P. stutzeri WM88 isolate. The DNA sequences of the minimal regions implicated in oxidn. of each compd. were detd. The region required for oxidn. of phosphite to phosphate putatively encodes a binding-protein-dependent phosphite transporter, an NAD+-dependent phosphite dehydrogenase, and a transcriptional activator of the lysR family. The region required for oxidn. of hypophosphite to phosphite putatively encodes a binding-protein-dependent hypophosphite transporter and an a-ketoglutarate-dependent hypophosphite dioxygenase. The finding of genes dedicated to oxidn. of reduced P compds. provides further evidence that a redox cycle for P may be important in the metab. of this essential, and often growth-limiting, nutrient.
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  82. 82
    Polyviou, D.; Hitchcock, A.; Baylay, A. J.; Moore, C. M.; Bibby, T. S. Phosphite Utilization by the Globally Important Marine Diazotroph Trichodesmium. Environ. Microbiol. Rep. 2015, 7 (6), 824– 830,  DOI: 10.1111/1758-2229.12308
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    Phosphite utilization by the globally important marine diazotroph Trichodesmium
    Polyviou, Despo; Hitchcock, Andrew; Baylay, Alison J.; Moore, C. Mark; Bibby, Thomas S.
    Environmental Microbiology Reports (2015), 7 (6), 824-830CODEN: EMRNAG; ISSN:1758-2229. (Wiley-Blackwell)
    Summary : Species belonging to the filamentous cyanobacterial genus Trichodesmium are responsible for a significant fraction of oceanic nitrogen fixation. The availability of phosphorus (P) likely constrains the growth of Trichodesmium in certain regions of the ocean. Moreover, Trichodesmium species have recently been shown to play a role in an emerging oceanic phosphorus redox cycle, further highlighting the key role these microbes play in many biogeochem. processes in the contemporary ocean. Here, we show that Trichodesmium erythraeum IMS101 can grow on the reduced inorg. compd. phosphite as its sole source of P. The components responsible for phosphite utilization are identified through heterologous expression of the T. erythraeum IMS101 Tery_0365-0368 genes, encoding a putative ATP (ATP)-binding cassette transporter and NAD (NAD)-dependent dehydrogenase, in the model cyanobacteria Synechocystis sp. PCC6803. We demonstrate that only combined expression of both the transporter and the dehydrogenase enables Synechocystis to utilize phosphite, confirming the function of Tery_0365-0367 as a phosphite uptake system (PtxABC) and Tery_0368 as a phosphite dehydrogenase (PtxD). Our findings suggest that reported uptake of phosphite by Trichodesmium consortia in the field likely reflects an active biol. process by Trichodesmium. These results highlight the diversity of phosphorus sources available to Trichodesmium in a resource-limited ocean.
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    Feingersch, R.; Philosof, A.; Mejuch, T.; Glaser, F.; Alalouf, O.; Shoham, Y.; Béjà, O. Potential for Phosphite and Phosphonate Utilization by Prochlorococcus. ISME J. 2012, 6 (4), 827– 834,  DOI: 10.1038/ismej.2011.149
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    Potential for phosphite and phosphonate utilization by Prochlorococcus
    Feingersch, Roi; Philosof, Alon; Mejuch, Tom; Glaser, Fabian; Alalouf, Onit; Shoham, Yuval; Beja, Oded
    ISME Journal (2012), 6 (4), 827-834CODEN: IJSOCF; ISSN:1751-7362. (Nature Publishing Group)
    Phosphonates (Pn) are diverse org. phosphorus (P) compds. contg. C-P bonds and comprise up to 25% of the high-mol. wt. dissolved org. P pool in the open ocean. Pn bioavailability was suggested to influence markedly bacterial primary prodn. in low-P areas. Using metagenomic data from the Global Ocean Sampling expedition, we show that the main potential microbial contributor in Pn utilization in oceanic surface water is the globally important marine primary producer Prochlorococcus. Moreover, a no. of Prochlorococcus strains contain two distinct putative Pn uptake operons coding for ABC-type Pn transporters. On the basis of microcalorimetric measurements, we find that each of the two different putative Pn-binding protein (PhnD) homologs transcribed from these operons possesses different Pn- as well as inorg. phosphite-binding specificities. Our results suggest that Prochlorococcus adapt to low-P environments by increasing the no. of Pn transporters with different specificities towards phosphite and different Pns.
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    Bisson, C.; Adams, N. B. P.; Stevenson, B.; Brindley, A. A.; Polyviou, D.; Bibby, T. S.; Baker, P. J.; Hunter, C. N.; Hitchcock, A. The Molecular Basis of Phosphite and Hypophosphite Recognition by ABC-Transporters. Nat. Commun. 2017, 8 (1), 1– 12,  DOI: 10.1038/s41467-017-01226-8
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    The molecular basis of phosphite and hypophosphite recognition by ABC-transporters
    Bisson, Claudine; Adams, Nathan B. P.; Stevenson, Ben; Brindley, Amanda A.; Polyviou, Despo; Bibby, Thomas S.; Baker, Patrick J.; Hunter, C. Neil; Hitchcock, Andrew
    Nature Communications (2017), 8 (1), 1-13CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Inorg. phosphate is the major bioavailable form of the essential nutrient phosphorus. However, the concn. of phosphate in most natural habitats is low enough to limit microbial growth. Under phosphate-depleted conditions some bacteria utilize phosphite and hypophosphite as alternative sources of phosphorus, but the mol. basis of reduced phosphorus acquisition from the environment is not fully understood. Here, we present crystal structures and ligand binding affinities of periplasmic binding proteins from bacterial phosphite and hypophosphite ATP-binding cassette transporters. We reveal that phosphite and hypophosphite specificity results from a combination of steric selection and the presence of a P-H...π interaction between the ligand and a conserved arom. residue in the ligand-binding pocket. The characterization of high affinity and specific transporters has implications for the marine phosphorus redox cycle, and might aid the use of phosphite as an alternative phosphorus source in biotechnol., industrial and agricultural applications.
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    Jumper, J. Highly Accurate Protein Structure Prediction with AlphaFold. Nature 2021, 596 (7873), 583– 589,  DOI: 10.1038/s41586-021-03819-2
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    Highly accurate protein structure prediction with AlphaFold
    Jumper, John; Evans, Richard; Pritzel, Alexander; Green, Tim; Figurnov, Michael; Ronneberger, Olaf; Tunyasuvunakool, Kathryn; Bates, Russ; Zidek, Augustin; Potapenko, Anna; Bridgland, Alex; Meyer, Clemens; Kohl, Simon A. A.; Ballard, Andrew J.; Cowie, Andrew; Romera-Paredes, Bernardino; Nikolov, Stanislav; Jain, Rishub; Adler, Jonas; Back, Trevor; Petersen, Stig; Reiman, David; Clancy, Ellen; Zielinski, Michal; Steinegger, Martin; Pacholska, Michalina; Berghammer, Tamas; Bodenstein, Sebastian; Silver, David; Vinyals, Oriol; Senior, Andrew W.; Kavukcuoglu, Koray; Kohli, Pushmeet; Hassabis, Demis
    Nature (London, United Kingdom) (2021), 596 (7873), 583-589CODEN: NATUAS; ISSN:0028-0836. (Nature Portfolio)
    Proteins are essential to life, and understanding their structure can facilitate a mechanistic understanding of their function. Through an enormous exptl. effort, the structures of around 100,000 unique proteins have been detd., but this represents a small fraction of the billions of known protein sequences. Structural coverage is bottlenecked by the months to years of painstaking effort required to det. a single protein structure. Accurate computational approaches are needed to address this gap and to enable large-scale structural bioinformatics. Predicting the three-dimensional structure that a protein will adopt based solely on its amino acid sequence-the structure prediction component of the 'protein folding problem'-has been an important open research problem for more than 50 years. Despite recent progress, existing methods fall far short of at. accuracy, esp. when no homologous structure is available. Here we provide the first computational method that can regularly predict protein structures with at. accuracy even in cases in which no similar structure is known. We validated an entirely redesigned version of our neural network-based model, AlphaFold, in the challenging 14th Crit. Assessment of protein Structure Prediction (CASP14), demonstrating accuracy competitive with exptl. structures in a majority of cases and greatly outperforming other methods. Underpinning the latest version of AlphaFold is a novel machine learning approach that incorporates phys. and biol. knowledge about protein structure, leveraging multi-sequence alignments, into the design of the deep learning algorithm.
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    Gupta, R.; Laxman, S. Cycles, Sources, and Sinks: Conceptualizing How Phosphate Balance Modulates Carbon Flux Using Yeast Metabolic Networks. Elife 2021, 10, e63341,  DOI: 10.7554/eLife.63341
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    Cycles, sources, and sinks: conceptualizing how phosphate balance modulates carbon flux using yeast metabolic networks
    Gupta, Ritu; Laxman, Sunil
    eLife (2021), 10 (), e63341CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
    Phosphates are ubiquitous mols. that enable crit. intracellular biochem. reactions. Therefore, cells have elaborate responses to phosphate limitation. Our understanding of long-term transcriptional responses to phosphate limitation is extensive. Contrastingly, a systemslevel perspective presenting unifying biochem. concepts to interpret how phosphate balance is critically coupled to (and controls) metabolic information flow is missing. To conceptualize such processes, utilizing yeast metabolic networks we categorize phosphates utilized in metab. into cycles, sources and sinks. Through this, we identify metabolic reactions leading to putative phosphate sources or sinks. With this conceptualization, we illustrate how mass action driven flux towards sources and sinks enable cells to manage phosphate availability during transient/ immediate phosphate limitations. We thereby identify how intracellular phosphate availability will predictably alter specific nodes in carbon metab., and det. signature cellular metabolic states. Finally, we identify a need to understand intracellular phosphate pools, in order to address mechanisms of phosphate regulation and restoration.
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    Simeonova, D. D.; Wilson, M. M.; Metcalf, W. W.; Schink, B. Identification and Heterologous Expression of Genes Involved in Anaerobic Dissimilatory Phosphite Oxidation by Desulfotignum Phosphitoxidans. J. Bacteriol. 2010, 192 (19), 5237– 5244,  DOI: 10.1128/JB.00541-10
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    87
    Identification and heterologous expression of genes involved in anaerobic dissimilatory phosphite oxidation by Desulfotignum phosphitoxidans
    Simeonova, Diliana Dancheva; Wilson, Marlena Marie; Metcalf, William W.; Schink, Bernhard
    Journal of Bacteriology (2010), 192 (19), 5237-5244CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
    Desulfotignum phosphitoxidans is a strictly anaerobic, Gram-neg. bacterium that utilizes phosphite as the sole electron source for homoacetogenic CO2 redn. or sulfate redn. A genomic library of D. phosphitoxidans, constructed using the fosmid vector pJK050, was screened for clones harboring the genes involved in phosphite oxidn. via PCR using primers developed based on the amino acid sequences of phosphite-induced proteins. Sequence anal. of two pos. clones revealed a putative operon of seven genes predicted to be involved in phosphite oxidn. Four of these genes (ptxD-ptdFCG) were cloned and heterologously expressed in Desulfotignum balticum, a related strain that cannot use phosphite as either an electron donor or as a phosphorus source. The ptxD-ptdFCG gene cluster was sufficient to confer phosphite uptake and oxidn. ability to the D. balticum host strain but did not allow use of phosphite as an electron donor for chemolithotrophic growth. Phosphite oxidn. activity was measured in cell exts. of D. balticum transconjugants, suggesting that all genes required for phosphite oxidn. were cloned. Genes of the phosphite gene cluster were assigned putative functions on the basis of sequence anal. and enzyme assays.
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  88. 88
    van Veen, H. W.; Abee, T.; Kortstee, G. J. J.; Konings, W. N.; Zehnder, A. J. B. Translocation of Metal Phosphate via the Phosphate Inorganic Transport System of Escherichia coli. Biochemistry 1994, 33 (7), 1766– 1770,  DOI: 10.1021/bi00173a020
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    88
    Translocation of Metal phosphate via the Phosphate Inorganic Transport System of Escherichia coli
    van Veen, Hendrik W.; Abee, Tjakko; Kortstee, Gerard J. J.; Konings, Wil N.; Zehnder, Alexander J. B.
    Biochemistry (1994), 33 (7), 1766-70CODEN: BICHAW; ISSN:0006-2960.
    Pi transport via the phosphate inorg. transport system (Pit) of E. coli was studied in natural and artificial membranes. Pi uptake via Pit is dependent on the presence of divalent cations, like Mg2+, Ca2+, Co2+, or Mn2+, which form a sol., neutral metal phosphate (MeHPO4) complex. Pi-dependent uptake of Mg2+ and Ca2+, equimolar cotransport of Pi and Ca2+, and inhibition by Mg2+ of Ca2+ uptake in the presence of Pi, but not of Pi uptake in the presence of Ca2+, indicate that a metal phosphate complex is the transported solute. Metal phosphate is transported in symport with H+ with a mechanistic stoichiometry of 1. Pit mediates efflux and homologous exchange of metal phosphate, but not heterologous metal phosphate exchange with Pi, glycerol-3P, or glucose-6P. The metal phosphate efflux rate increased with pH, whereas the rate of metal phosphate exchange was essentially pH independent. Metal phosphate uptake was inhibited at low internal pH. Efflux was inhibited by a proton motive force (interior neg. and alk.), whereas exchange was inhibited by the membrane potential only. These results have been evaluated in terms of ordered binding and dissocn. of metal phosphate and protons on the outer and inner surface of the cytoplasmic membrane.
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  89. 89
    Pedersen, B. P.; Kumar, H.; Waight, A. B.; Risenmay, A. J.; Roe-Zurz, Z.; Chau, B. H.; Schlessinger, A.; Bonomi, M.; Harries, W.; Sali, A.; Johri, A. K.; Stroud, R. M. Crystal Structure of a Eukaryotic Phosphate Transporter. Nature 2013, 496 (7446), 533– 536,  DOI: 10.1038/nature12042
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    89
    Crystal structure of a eukaryotic phosphate transporter
    Pedersen, Bjorn P.; Kumar, Hemant; Waight, Andrew B.; Risenmay, Aaron J.; Roe-Zurz, Zygy; Chau, Bryant H.; Schlessinger, Avner; Bonomi, Massimiliano; Harries, William; Sali, Andrej; Johri, Atul K.; Stroud, Robert M.
    Nature (London, United Kingdom) (2013), 496 (7446), 533-536CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Phosphate is crucial for structural and metabolic needs, including nucleotide and lipid synthesis, signalling and chem. energy storage. Proton-coupled transporters of the major facilitator superfamily (MFS) are essential for phosphate uptake in plants and fungi, and also have a function in sensing external phosphate levels as transceptors. Here we report the 2.9 Å structure of a fungal (Piriformospora indica) high-affinity phosphate transporter, PiPT, in an inward-facing occluded state, with bound phosphate visible in the membrane-buried binding site. The structure indicates both proton and phosphate exit pathways and suggests a modified asym. 'rocker-switch' mechanism of phosphate transport. PiPT is related to several human transporter families, most notably the org. cation and anion transporters of the solute carrier family (SLC22), which are implicated in cancer-drug resistance. We modelled representative cation and anion SLC22 transporters based on the PiPT structure to surmise the structural basis for substrate binding and charge selectivity in this important family. The PiPT structure demonstrates and expands on principles of substrate transport by the MFS transporters and illuminates principles of phosphate uptake in particular.
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  90. 90
    de Pinto, V.; Tommasino, M.; Palmieri, F.; Kadenbach, B. Purification of the Active Mitochondrial Phosphate Carrier by Affinity Chromatography with an Organomercurial Agarose Column. FEBS Lett. 1982, 148 (1), 103– 106,  DOI: 10.1016/0014-5793(82)81252-0
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    90
    Purification of the active mitochondrial phosphate carrier by affinity chromatography with an organomercurial agarose column
    De Pinto, V.; Tommasino, M.; Palmieri, F.; Kadenbach, B.
    FEBS Letters (1982), 148 (1), 103-6CODEN: FEBLAL; ISSN:0014-5793.
    Active phosphate carrier was purified by affinity chromatog. from a hydroxylapatite eluate of Triton X 100-solubilized mitochondria. Affinity chromatog. was performed with Affi-Gel 501 as stationary phase and with a mercaptoethanol gradient in the mobile phase. One main protein band was obsd. after high-resoln. SDS gel electrophoresis of this prepn.
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  91. 91
    Motomura, K.; Hirota, R.; Ohnaka, N.; Okada, M.; Ikeda, T.; Morohoshi, T.; Ohtake, H.; Kuroda, A. Overproduction of YjbB Reduces the Level of Polyphosphate in Escherichia coli: A Hypothetical Role of YjbB in Phosphate Export and Polyphosphate Accumulation. FEMS Microbiol. Lett. 2011, 320 (1), 25– 32,  DOI: 10.1111/j.1574-6968.2011.02285.x
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    91
    Overproduction of YjbB reduces the level of polyphosphate in Escherichia coli: a hypothetical role of YjbB in phosphate export and polyphosphate accumulation
    Motomura, Kei; Hirota, Ryuichi; Ohnaka, Nobuteru; Okada, Mai; Ikeda, Takeshi; Morohoshi, Tomohiro; Ohtake, Hisao; Kuroda, Akio
    FEMS Microbiology Letters (2011), 320 (1), 25-32CODEN: FMLED7; ISSN:0378-1097. (Wiley-Blackwell)
    Intracellular phosphate (Pi) is normally maintained at a fairly const. concn. in Escherichia coli, mainly by Pi transport systems and by the "phosphate balance" between Pi and polyphosphate (polyP). We have reported previously that excess uptake of Pi in a phoU mutant results in elevated levels of polyP. Here, we found that the elevated levels of polyP in the mutant could be reduced by the overprodn. of YjbB, whose N-terminal half contains Na+/Pi cotransporter domains. The rate of Pi export increased when the YjbB overproducer grew on a medium contg. glycerol-3-phosphate. These results strongly suggested that YjbB reduced the elevated levels of polyP in the phoU mutant by exporting intracellular excess Pi.
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  92. 92
    Hamburger, D.; Rezzonico, E.; MacDonald-Comber Petetot, J.; Somerville, C.; Poirier, Y. Identification and Characterization of the Arabidopsis PHO1 Gene Involved in Phosphate Loading to the Xylem. Plant Cell 2002, 14 (4), 889– 902,  DOI: 10.1105/tpc.000745
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    92
    Identification and characterization of the Arabidopsis PHO1 gene involved in phosphate loading to the xylem
    Hamburger, Dirk; Rezzonico, Enea; Petetot, Jean MacDonald-Comber; Somerville, Chris; Poirier, Yves
    Plant Cell (2002), 14 (4), 889-902CODEN: PLCEEW; ISSN:1040-4651. (American Society of Plant Biologists)
    The Arabidopsis mutant pho1 is deficient in the transfer of Pi from root epidermal and cortical cells to the xylem. The PHO1 gene was identified by a map-based cloning strategy. The N-terminal half of PHO1 is mainly hydrophilic, whereas the C-terminal half has six potential membrane-spanning domains. PHO1 shows no homol. with any characterized solute transporter, including the family of H+-Pi cotransporters identified in plants and fungi. PHO1 shows highest homol. with the Rcm1 mammalian receptor for xenotropic murine leukemia retroviruses and with the Saccharomyces cerevisiae Syg1 protein involved in the mating pheromone signal transduction pathway. PHO1 is expressed predominantly in the roots and is upregulated weakly under Pi stress. Studies with PHO1 promoter-β-glucuronidase constructs reveal predominant expression of the PHO1 promoter in the stelar cells of the root and the lower part of the hypocotyl. There also is β-glucuronidase staining of endodermal cells that are adjacent to the protoxylem vessels. The Arabidopsis genome contains 10 addnl. genes showing homol. with PHO1. Thus, PHO1 defines a novel class of proteins involved in ion transport in plants.
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  93. 93
    Palmieri, F.; Indiveri, C.; Bisaccia, F.; Krämer, R. Functional Properties of Purified and Reconstituted Mitochondrial Metabolite Carriers. J. Bioenerg. Biomembr. 1993, 25 (5), 525– 535,  DOI: 10.1007/BF01108409
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    93
    Functional properties of purified and reconstituted mitochondrial metabolite carriers
    Palmieri, F.; Indiveri, C.; Bisaccia, F.; Kraemer, R.
    Journal of Bioenergetics and Biomembranes (1993), 25 (5), 525-35CODEN: JBBID4; ISSN:0145-479X.
    A review with ∼60 refs. Eight mitochondrial carrier proteins were solubilized and purified in the authors' labs. using variations of a general procedure based on hydroxyapatite and Celite chromatog. The mol. mass of all the carriers ranges between 28 and 34 kDa on SDS-PAGE. The purified carrier proteins were reconstituted into liposomes mainly by using a method of detergent removal by hydrophobic chromatog. on polystyrene beads. The various carriers were identified in the reconstituted state by their kinetic properties. A complete set of basic kinetic data including substrate specificity, affinity, interaction with inhibitors, and activation energy was obtained. These data closely resemble those of intact mitochondria, as far as they are available from the intact organelle. Mainly on the basis of kinetic data, the asym. orientation of most of the reconstituted carrier proteins were established. Several of their functional properties are significantly affected by the type of phospholipids used for reconstitution.
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  94. 94
    Seddon, A. M.; Curnow, P.; Booth, P. J. Membrane Proteins, Lipids and Detergents: Not Just a Soap Opera. Biochim. Biophys. Acta - Biomembr. 2004, 1666 (1–2), 105– 117,  DOI: 10.1016/j.bbamem.2004.04.011
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    94
    Membrane proteins, lipids and detergents: not just a soap opera
    Seddon, Annela M.; Curnow, Paul; Booth, Paula J.
    Biochimica et Biophysica Acta, Biomembranes (2004), 1666 (1-2), 105-117CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
    A review. Studying membrane proteins represents a major challenge in protein biochem., with one of the major difficulties being the problems encountered when working outside the natural lipid environment. In vitro studies such as crystn. are reliant on the successful solubilization or reconstitution of membrane proteins, which generally involves the careful selection of solubilizing detergents and mixed lipid/detergent systems. This review will conc. on the methods currently available for efficient reconstitution and solubilization of membrane proteins through the use of detergent micelles, mixed lipid/detergent micelles and bicelles or liposomes. We focus on the relevant mol. properties of the detergents and lipids that aid understanding of these processes. A significant barrier to membrane protein research is retaining the stability and function of the protein during solubilization, reconstitution and crystn. We highlight some of the lessons learnt from studies of membrane protein folding in vitro and give an overview of the role that lipids can play in stabilizing the proteins.
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  95. 95
    Junge, F.; Schneider, B.; Reckel, S.; Schwarz, D.; Dötsch, V.; Bernhard, F. Large-Scale Production of Functional Membrane Proteins. Cell. Mol. Life Sci. 2008, 65 (11), 1729– 1755,  DOI: 10.1007/s00018-008-8067-5
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    95
    Large-scale production of functional membrane proteins
    Junge, F.; Schneider, B.; Reckel, S.; Schwarz, D.; Doetsch, V.; Bernhard, F.
    Cellular and Molecular Life Sciences (2008), 65 (11), 1729-1755CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Verlag)
    A review. The prepn. of sufficient amts. of high-quality samples is still the major bottleneck for the characterization of membrane proteins by in vitro approaches. The hydrophobic nature, the requirement for complicated transport and modification pathways, and the often obsd. neg. effects on membrane properties are intrinsic features of membrane proteins that frequently cause significant problems in overexpression studies. Establishing efficient protocols for the prodn. of functionally folded membrane proteins is therefore a challenging task, and numerous specific characteristics have to be considered. In addn., a variety of expression systems have been developed, and choice of appropriate techniques could strongly depend on the desired target membrane proteins as well as on their intended applications. The prodn. of membrane proteins is a highly dynamic field and new or modified approaches are frequently emerging. The review will give an overview of currently established processes for the prodn. of functionally folded membrane proteins.
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  96. 96
    Amati, A. M.; Graf, S.; Deutschmann, S.; Dolder, N.; von Ballmoos, C. Current Problems and Future Avenues in Proteoliposome Research. Biochem. Soc. Trans. 2020, 48 (4), 1473– 1492,  DOI: 10.1042/BST20190966
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    96
    Current problems and future avenues in proteoliposome research
    Amati, Andrea Marco; Graf, Simone; Deutschmann, Sabina; Dolder, Nicolas; von Ballmoos, Christoph
    Biochemical Society Transactions (2020), 48 (4), 1473-1492CODEN: BCSTB5; ISSN:0300-5127. (Portland Press Ltd.)
    A review. Membrane proteins (MPs) are the gatekeepers between different biol. compartments sepd. by lipid bilayers. Being receptors, channels, transporters, or primary pumps, they fulfill a wide variety of cellular functions and their importance is reflected in the increasing no. of drugs that target MPs. Functional studies of MPs within a native cellular context, however, is difficult due to the innate complexity of the densely packed membranes. Over the past decades, detergent-based extn. and purifn. of MPs and their reconstitution into lipid mimetic systems has been a very powerful tool to simplify the exptl. system. In this review, we focus on proteoliposomes that have become an indispensable exptl. system for enzymes with a vectorial function, including many of the here described energy transducing MPs. We first address long standing questions on the difficulty of successful reconstitution and controlled orientation of MPs into liposomes. A special emphasis is given on coreconstitution of several MPs into the same bilayer. Second, we discuss recent progress in the development of fluorescent dyes that offer sensitive detection with high temporal resoln. Finally, we briefly cover the use of giant unilamellar vesicles for the investigation of complex enzymic cascades, a very promising exptl. tool considering our increasing knowledge of the interplay of different cellular components.
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  97. 97
    Warnecke, T.; Gill, R. T. Organic Acid Toxicity, Tolerance, and Production in Escherichia coli Biorefining Applications. Microb. Cell Fact. 2005, 4 (1), 1– 8,  DOI: 10.1186/1475-2859-4-25
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    Voskuhl, L.; Brusilova, D.; Brauer, V. S.; Meckenstock, R. U. Inhibition of Sulfate-Reducing Bacteria with Formate. FEMS Microbiol. Ecol. 2022, 98 (1), 1– 10,  DOI: 10.1093/femsec/fiac003
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  99. 99
    Gabba, M.; Frallicciardi, J.; van ’t Klooster, J.; Henderson, R.; Syga, Ł.; Mans, R.; van Maris, A. J. A.; Poolman, B. Weak Acid Permeation in Synthetic Lipid Vesicles and Across the Yeast Plasma Membrane. Biophys. J. 2020, 118 (2), 422– 434,  DOI: 10.1016/j.bpj.2019.11.3384
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    99
    Weak Acid Permeation in Synthetic Lipid Vesicles and Across the Yeast Plasma Membrane
    Gabba, Matteo; Frallicciardi, Jacopo; van 't Klooster, Joury; Henderson, Ryan; Syga, Lukasz; Mans, Robert; van Maris, Antonius J. A.; Poolman, Bert
    Biophysical Journal (2020), 118 (2), 422-434CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    The authors present a fluorescence-based approach for detn. of the permeability of small mols. across the membranes of lipid vesicles and living cells. With properly designed expts., the method allows the authors to assess the membrane phys. properties both in vitro and in vivo. The permeability of weak acids increases in the order of benzoic > acetic > formic > lactic, both in synthetic lipid vesicles and the plasma membrane of Saccharomyces cerevisiae, but the permeability is much lower in yeast (one to two orders of magnitude). A relation between the mol. permeability and the satn. of the lipid acyl chain (i.e., lipid packing) in the synthetic lipid vesicles. were obsd. By analyzing wild-type yeast and a manifold knockout strain lacking all putative lactic acid transporters, the yeast plasma membrane is impermeable to lactic acid on timescales up to ∼2.5 h.
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  100. 100
    Amao, Y. Formate Dehydrogenase for CO2 Utilization and Its Application. J. CO2 Util. 2018, 26, 623– 641,  DOI: 10.1016/j.jcou.2018.06.022
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    100
    Formate dehydrogenase for CO2 utilization and its application
    Amao, Yutaka
    Journal of CO2 Utilization (2018), 26 (), 623-641CODEN: JCUOAJ; ISSN:2212-9839. (Elsevier Ltd.)
    Carbon dioxide, CO2 redn. and utilization for org. compds. synthesis are the potential technologies in environmental science and technol. In order to establish efficient CO2 utilization technologies, an effective catalyst for CO2 redn. and utilization is necessary. Among various catalysts, the biocatalyst is one of promising catalysts because it has excellent selectivity for the reaction and substrate. In this review, focusing on biocatalyst "formate dehydrogenase FDH" catalyzing CO2 redn. to formic acid, representative examples of properties, types, structure of active-site of FDH and, reaction mechanism of formic acid oxidn. and CO2 redn. with FDH are outlined. A genetic engineering modified FDH and FDH immobilized various support for improving CO2 redn. catalytic activity also are introduced. Moreover, chem. and electrochem. system of CO2 redn. to formic acid with FDH, aq. homogenous system of visible-light driven CO2 redn. to formic acid with FDH and device for visible-light driven CO2 redn. to formic acid with FDH are also introduced as an application of FDH.
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  101. 101
    Maia, L. B.; Moura, I.; Moura, J. J. G. Molybdenum and Tungsten-Containing Formate Dehydrogenases: Aiming to Inspire a Catalyst for Carbon Dioxide Utilization. Inorg. Chim. Acta 2017, 455, 350– 363,  DOI: 10.1016/j.ica.2016.07.010
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    101
    Molybdenum and tungsten-containing formate dehydrogenases: Aiming to inspire a catalyst for carbon dioxide utilization
    Maia, Luisa B.; Moura, Isabel; Moura, Jose J. G.
    Inorganica Chimica Acta (2017), 455 (Part_2), 350-363CODEN: ICHAA3; ISSN:0020-1693. (Elsevier B.V.)
    A review concerning the use Mo- and W-contg. formate dehydrogenase (FDH) enzymes as a model to understand mechanistic strategies and key chem. features needed to reduce CO2 to formate, highlighting current knowledge about the FDH structure, emphasizing active site features, reaction mechanism, and ability to reduce CO2 to formate, is given. The gathered information aims to inspire development of new efficient bio-catalysts for atm. CO2 utilization to produce energy and chem. feedstocks while reducing an important environmental pollutant. Topics covered include: the CO2 crisis; FDH enzymes (families, Mo- and W-contg. FDH, mechanistic strategies for FDH, new look at FDH catalysis); FDH-catalyzed CO2 redn.; and outlook.
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  102. 102
    Tishkov, V. I.; Matorin, A. D.; Rojkova, A. M.; Fedorchuk, V. V.; Savitsky, P. A.; Dementieva, L. A.; Lamzin, V. S.; Mezentzev, A. V.; Popov, V. O. Site-Directed Mutagenesis of the Formate Dehydrogenase Active Centre: Role of the His332-Gln313 Pair in Enzyme Catalysis. FEBS Lett. 1996, 390 (1), 104– 108,  DOI: 10.1016/0014-5793(96)00641-2
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    102
    Site-directed mutagenesis of the formate dehydrogenase active center: role of the His332-Gln313 pair in enzyme catalysis
    Tishkov, Vladimir I.; Matorin, Andrey D.; Rojkova, Alexandra M.; Fedorchuk, Vladimir V.; Savitsky, Pavel A.; Dementieva, Larissa A.; Lamzin, Victor S.; Mezentzev, Alexander V.; Popov, Vladimir O.
    FEBS Letters (1996), 390 (1), 104-108CODEN: FEBLAL; ISSN:0014-5793. (Elsevier)
    Gln313 and His332 residues in the active center of NAD+-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from the bacterium Pseudomonas sp. 101 are conserved in all FDHs and are equiv. to the glutamate-histidine pair in active sites of D-specific 2-hydroxy acid dehydrogenases. Two mutants of formate dehydrogenase from Pseudomonas sp. 101, Gln313Glu and His332Phe, have been obtained and characterized. The Gln313Glu mutation shifts the pK of the group controlling formate binding from less than 5.5 in wild-type enzyme to 7.6 thus indicating that Gln313 is essential for the broad pH affinity profile towards substrate. His332Phe mutation leads to a complete loss of enzyme activity. The His332Phe mutant is still able to bind coenzyme but not substrate or analogs. The role of histidine in the active center of FDH is discussed. The protonation state of His332 is not crit. for catalysis but vital for substrate binding. A partial pos. charge on the histidine imidazole, required for substrate binding, is provided via tight H-bond to the Gln313 carboxamide.
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  103. 103
    Lamzin, V. S.; Dauter, Z.; Popov, V. O.; Harutyunyan, E. H.; Wilson, K. S. High Resolution Structures of Holo and Apo Formate Dehydrogenase. J. Mol. Biol. 1994, 236, 759– 785,  DOI: 10.1006/jmbi.1994.1188
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    103
    High resolution structures of holo and apo formate dehydrogenase
    Lamzin, Victor S.; Dauter, Zbigniew; Popov, Vladimir O.; Harutyunyan, Emil H.; Wilson, Keith S.
    Journal of Molecular Biology (1994), 236 (3), 759-85CODEN: JMOBAK; ISSN:0022-2836.
    Three-dimensional crystal structures of holo (ternary complex enzyme-NAD-azide) and apo NAD-dependent dimeric formate dehydrogenase (FDH) from the methylotrophic bacterium Pseudomonas sp. 101 have been refined to R factors of 11.7% and 14.8% at 2.05 and 1.80 Å resoln., resp. The estd. root-mean-square error in at. coordinates is 0.11 Å for holo and 0.18 Å for apo. X-ray data were collected from single crystals using an imaging plate scanner and synchrotron radiation. In both crystal forms there is a dimer in the asym. unit. Both structures show essentially 2-fold mol. symmetry. NAD binding causes movement of the catalytic domain and ordering of the C terminus, where a new helix appears. This completes formation of the enzyme active center in holo FDH. NAD is bound in the cleft sepg. the domains and mainly interacts with residues from the co-enzyme binding domain. In apo FDH these residues are held in essentially the same conformation by water mols. occupying the NAD binding region. An azide mol. is located near the point of catalysis, the C4 atom of the nicotinamide moiety of NAD, and overlaps with the proposed formate binding site. There is an extensive channel running from the active site to the protein surface and this is supposed to be used by substrate to reach the active center after NAD has already bound. The structure of the active site and a hypothetical catalytic mechanism are discussed. Sequence homol. of FDH with other NAD-dependent formate dehydrogenases and some D-specific dehydrogenases is discussed on the basis of the FDH three-dimensional structure.
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  104. 104
    Hoelsch, K.; Sührer, I.; Heusel, M.; Weuster-Botz, D. Engineering of Formate Dehydrogenase: Synergistic Effect of Mutations Affecting Cofactor Specificity and Chemical Stability. Appl. Microbiol. Biotechnol. 2013, 97 (6), 2473– 2481,  DOI: 10.1007/s00253-012-4142-9
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    104
    Engineering of formate dehydrogenase: synergistic effect of mutations affecting cofactor specificity and chemical stability
    Hoelsch, Kathrin; Suehrer, Ilka; Heusel, Moritz; Weuster-Botz, Dirk
    Applied Microbiology and Biotechnology (2013), 97 (6), 2473-2481CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    Formate dehydrogenases (FDHs) are frequently used for the regeneration of cofactors in biotransformations employing NAD(P)H-dependent oxidoreductases. Major drawbacks of most native FDHs are their strong preference for NAD+ and their low operational stability in the presence of reactive org. compds. such as α-haloketones. In this study, the FDH from Mycobacterium vaccae N10 (MycFDH) was engineered in order to obtain an enzyme that is not only capable of regenerating NADPH but also stable toward the α-haloketone Et 4-chloroacetoacetate (ECAA). To change the cofactor specificity, amino acids in the conserved NAD+ binding motif were mutated. Among these mutants, MycFDH A198G/D221Q had the highest catalytic efficiency (kcat/K m) with NADP+. The addnl. replacement of two cysteines (C145S/C255V) not only conferred a high resistance to ECAA but also enhanced the catalytic efficiency 6-fold. The resulting quadruple mutant MycFDH C145S/A198G/D221Q/C255V had a specific activity of 4.00 ± 0.13 U mg-1 and a K of 0.147 ± 0.020 mM at 30 °C, pH 7. The A198G replacement had a major impact on the kinetic consts. of the enzyme. The corresponding triple mutant, MycFDH C145S/D221Q/C255V, showed the highest specific activity reported to date for a NADP+-accepting FDH (vmax, 10.25 ± 1.63 U mg-1). However, the half-satn. const. for NADP+ (Km, 0.92 ± 0.10 mM) was about one order of magnitude higher than the one of the quadruple mutant. Depending on the reaction setup, both novel MycFDH variants could be useful for the prodn. of the chiral synthon Et (S)-4-chloro-3-hydroxybutyrate [(S)-ECHB] by asym. redn. of ECAA with NADPH-dependent ketoreductases.
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  105. 105
    Calzadiaz-Ramirez, L.; Calvó-Tusell, C.; Stoffel, G. M. M.; Lindner, S. N.; Osuna, S.; Erb, T. J.; Garcia-Borràs, M.; Bar-Even, A.; Acevedo-Rocha, C. G. In Vivo Selection for Formate Dehydrogenases with High Efficiency and Specificity toward NADP+. ACS Catal. 2020, 10 (14), 7512– 7525,  DOI: 10.1021/acscatal.0c01487
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    In vivo selection for formate dehydrogenases with high efficiency and specificity toward NADP+
    Calzadiaz-Ramirez, Liliana; Calvo-Tusell, Carla; Stoffel, Gabriele M. M.; Lindner, Steffen N.; Osuna, Silvia; Erb, Tobias J.; Garcia-Borras, Marc; Bar-Even, Arren; Acevedo-Rocha, Carlos G.
    ACS Catalysis (2020), 10 (14), 7512-7525CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
    The efficient regeneration of cofactors is vital for the establishment of biocatalytic processes. Formate is an ideal electron donor for cofactor regeneration due to its general availability, low redn. potential, and benign byproduct (CO2). However, formate dehydrogenases (FDHs) are usually specific to NAD+, such that NADPH regeneration with formate is challenging. Previous studies reported naturally occurring FDHs or engineered FDHs that accept NADP+, but these enzymes show low kinetic efficiencies and specificities. Here, we harness the power of natural selection to engineer FDH variants to simultaneously optimize three properties: kinetic efficiency with NADP+, specificity toward NADP+, and affinity toward formate. By simultaneously mutating multiple residues of FDH from Pseudomonas sp. 101, which exhibits practically no activity toward NADP+, we generate a library of >106 variants. We introduce this library into an E. coli strain that cannot produce NADPH. By selecting for growth with formate as the sole NADPH source, we isolate several enzyme variants that support efficient NADPH regeneration. We find that the kinetically superior enzyme variant, harboring five mutations, has 5-fold higher efficiency and 14-fold higher specificity in comparison to the best enzyme previously engineered, while retaining high affinity toward formate. By using mol. dynamics simulations, we reveal the contribution of each mutation to the superior kinetics of this variant. We further det. how nonadditive epistatic effects improve multiple parameters simultaneously. Our work demonstrates the capacity of in vivo selection to identify highly proficient enzyme variants carrying multiple mutations which would be almost impossible to find using conventional screening methods.
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  106. 106
    Partipilo, M.; Whittaker, J.J.; Pontillo, N.; Coenradij, J.; Herrmann, A.; Guskov, A.; Slotboom, D. J. Biochemical and Structural Insight into the Chemical Resistance and Cofactor Specificity of the Formate Dehydrogenase from Starkeya Novella. FEBS J.
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    Endeward, V.; Al-Samir, S.; Itel, F.; Gros, G. How Does Carbon Dioxide Permeate Cell Membranes? A Discussion of Concepts, Results and Methods. Front. Physiol. 2014, 4, 382,  DOI: 10.3389/fphys.2013.00382
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    How does carbon dioxide permeate cell membranes? A discussion of concepts, results and methods
    Endeward Volker; Al-Samir Samer; Gros Gerolf; Itel Fabian
    Frontiers in physiology (2014), 4 (), 382 ISSN:1664-042X.
    We review briefly how the thinking about the permeation of gases, especially CO2, across cell and artificial lipid membranes has evolved during the last 100 years. We then describe how the recent finding of a drastic effect of cholesterol on CO2 permeability of both biological and artificial membranes fundamentally alters the long-standing idea that CO2-as well as other gases-permeates all membranes with great ease. This requires revision of the widely accepted paradigm that membranes never offer a serious diffusion resistance to CO2 or other gases. Earlier observations of "CO2-impermeable membranes" can now be explained by the high cholesterol content of some membranes. Thus, cholesterol is a membrane component that nature can use to adapt membrane CO2 permeability to the functional needs of the cell. Since cholesterol serves many other cellular functions, it cannot be reduced indefinitely. We show, however, that cells that possess a high metabolic rate and/or a high rate of O2 and CO2 exchange, do require very high CO2 permeabilities that may not be achievable merely by reduction of membrane cholesterol. The article then discusses the alternative possibility of raising the CO2 permeability of a membrane by incorporating protein CO2 channels. The highly controversial issue of gas and CO2 channels is systematically and critically reviewed. It is concluded that a majority of the results considered to be reliable, is in favor of the concept of existence and functional relevance of protein gas channels. The effect of intracellular carbonic anhydrase, which has recently been proposed as an alternative mechanism to a membrane CO2 channel, is analysed quantitatively and the idea considered untenable. After a brief review of the knowledge on permeation of O2 and NO through membranes, we present a summary of the (18)O method used to measure the CO2 permeability of membranes and discuss quantitatively critical questions that may be addressed to this method.
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  108. 108
    Lubitz, W.; Ogata, H.; Rudiger, O.; Reijerse, E. Hydrogenases. Chem. Rev. 2014, 114 (8), 4081– 4148,  DOI: 10.1021/cr4005814
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    Hydrogenases
    Lubitz, Wolfgang; Ogata, Hideaki; Ruediger, Olaf; Reijerse, Edward
    Chemical Reviews (Washington, DC, United States) (2014), 114 (8), 4081-4148CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. The current state of knowledge on hydrogenases, esp. recent advances made in understanding the detailed structure and function of these important enzymes. The authors provide an overview of important previous achievements with the main focus on [NiFe] and [FeFe] hydrogenases, and in part also on [Fe] hydrogenases. Recent progress on biomimetic model systems for hydrogenases and devices using hydrogenases both in fuel cells and for H2 prodn. are presented with emphasis on functional aspects. The great progress made in synthesizing model systems for hydrogenases that are functionally active is promising for the future employment of such catalysts in hydrogen technologies.
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  109. 109
    Lauterbach, L.; Lenz, O.; Vincent, K. A. H2-Driven Cofactor Regeneration with NAD(P)+-Reducing Hydrogenases. FEBS J. 2013, 280 (13), 3058– 3068,  DOI: 10.1111/febs.12245
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    H2-driven cofactor regeneration with NAD(P)+-reducing hydrogenases
    Lauterbach, Lars; Lenz, Oliver; Vincent, Kylie A.
    FEBS Journal (2013), 280 (13), 3058-3068CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
    A review. A large no. of industrially relevant enzymes depend upon nicotinamide cofactors, which are too expensive to be added in stoichiometric amts. Existing NAD(P)H-recycling systems suffer from low activity, or the generation of side products. H2-driven cofactor regeneration has the advantage of 100% atom efficiency and the use of H2 as a cheap reducing agent, in a world where sustainable energy carriers are increasingly attractive. The state of development of H2-driven cofactor-recycling systems and examples of their integration with enzyme reactions are summarized in this article. The O2-tolerant NAD+-reducing hydrogenase from Ralstonia eutropha is a particularly attractive candidate for this approach, and we therefore discuss its catalytic properties that are relevant for tech. applications.
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    Vandamme, P.; Coenye, T. Taxonomy of the Genus Cupriavidus: A Tale of Lost and Found. Int. J. Syst. Evol. Microbiol. 2004, 54 (6), 2285– 2289,  DOI: 10.1099/ijs.0.63247-0
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    Taxonomy of the genus Cupriavidus: a tale of lost and found
    Vandamme Peter; Coenye Tom
    International journal of systematic and evolutionary microbiology (2004), 54 (Pt 6), 2285-2289 ISSN:1466-5026.
    DNA-DNA hybridization experiments and an evaluation of phenotypic characteristics, DNA base ratios and 16S rRNA gene sequences demonstrated that Wautersia eutropha (Davies 1969) Vaneechoutte et al. 2004, the type species of the genus Wautersia, is a later synonym of Cupriavidus necator Makkar and Casida 1987, the type species of the genus Cupriavidus. In conformity with Rules 15, 17, 23a and 37a(1) of the International Code of Nomenclature of Bacteria, the genus name Cupriavidus has priority over the genus name Wautersia, and all other members of the genus Wautersia are reclassified into Cupriavidus as Cupriavidus basilensis comb. nov. (type strain LMG 18990(T)=DSM 11853(T)), Cupriavidus campinensis comb. nov. (type strain LMG 19282(T)=CCUG 44526(T)), Cupriavidus gilardii comb. nov. (type strain LMG 5886(T)=CCUG 38401(T)), Cupriavidus metallidurans comb. nov. (type strain LMG 1195(T)=DSM 2839(T)), Cupriavidus oxalaticus comb. nov. (type strain LMG 2235(T)=CCUG 2086(T)=DSM 1105(T)), Cupriavidus pauculus comb. nov. (type strain LMG 3244(T)=CCUG 12507(T)), Cupriavidus respiraculi comb. nov. (type strain LMG 21510(T)=CCUG 46809(T)) and Cupriavidus taiwanensis comb. nov. (type strain LMG 19424(T)=CCUG 44338(T)).
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  111. 111
    Shomura, Y.; Taketa, M.; Nakashima, H.; Tai, H.; Nakagawa, H.; Ikeda, Y.; Ishii, M.; Igarashi, Y.; Nishihara, H.; Yoon, K. S.; Ogo, S.; Hirota, S.; Higuchi, Y. Structural Basis of the Redox Switches in the NAD+-Reducing Soluble [NiFe]-Hydrogenase. Science. 2017, 357 (6354), 928– 932,  DOI: 10.1126/science.aan4497
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    Structural basis of the redox switches in the NAD+-reducing soluble [NiFe]-hydrogenase
    Shomura, Y.; Taketa, M.; Nakashima, H.; Tai, H.; Nakagawa, H.; Ikeda, Y.; Ishii, M.; Igarashi, Y.; Nishihara, H.; Yoon, K.-S.; Ogo, S.; Hirota, S.; Higuchi, Y.
    Science (Washington, DC, United States) (2017), 357 (6354), 928-932CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    NAD-reducing sol. [NiFe]-hydrogenase (SH) is phylogenetically related to NADH:quinone oxidoreductase (complex I), but the geometrical arrangements of the subunits and Fe-S clusters are unclear. Here, we describe the crystal structures of SH of Hydrogenophilus thermoluteolus in the oxidized and reduced states. The cluster arrangement was similar to that of complex I, but the subunits orientation was not, which supported the hypothesis that subunits evolved as prebuilt modules. The oxidized active site included a 6-coordinate Ni, which was unprecedented for hydrogenases, whose coordination geometry would prevent O2 from approaching. In the reduced state showing the normal active site structure without a physiol. electron acceptor, the FMN cofactor was dissocd., which may be caused by the oxidn. state change of nearby Fe-S clusters and may suppress the prodn. of reactive oxygen species.
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  112. 112
    Kulka-Peschke, C. J.; Schulz, A. C.; Lorent, C.; Rippers, Y.; Wahlefeld, S.; Preissler, J.; Schulz, C.; Wiemann, C.; Bernitzky, C. C. M.; Karafoulidi-Retsou, C.; Wrathall, S. L. D.; Procacci, B.; Matsuura, H.; Greetham, G. M.; Teutloff, C.; Lauterbach, L.; Higuchi, Y.; Ishii, M.; Hunt, N. T.; Lenz, O.; Zebger, I.; Horch, M. Reversible Glutamate Coordination to High-Valent Nickel Protects the Active Site of a [NiFe] Hydrogenase from Oxygen. J. Am. Chem. Soc. 2022, 144 (37), 17022– 17032,  DOI: 10.1021/jacs.2c06400
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    112
    Reversible Glutamate Coordination to High-Valent Nickel Protects the Active Site of a [NiFe] Hydrogenase from Oxygen
    Kulka-Peschke, Catharina J.; Schulz, Anne-Christine; Lorent, Christian; Rippers, Yvonne; Wahlefeld, Stefan; Preissler, Janina; Schulz, Claudia; Wiemann, Charlotte; Bernitzky, Cornelius C. M.; Karafoulidi-Retsou, Chara; Wrathall, Solomon L. D.; Procacci, Barbara; Matsuura, Hiroaki; Greetham, Gregory M.; Teutloff, Christian; Lauterbach, Lars; Higuchi, Yoshiki; Ishii, Masaharu; Hunt, Neil T.; Lenz, Oliver; Zebger, Ingo; Horch, Marius
    Journal of the American Chemical Society (2022), 144 (37), 17022-17032CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    NAD+-reducing [NiFe] hydrogenases are valuable biocatalysts for H2-based energy conversion and the regeneration of nucleotide cofactors. While most hydrogenases are sensitive toward O2 and elevated temps., the sol. NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus (HtSH) is O2-tolerant and thermostable. Thus, it represents a promising candidate for biotechnol. applications. Here, we have investigated the catalytic activity and active-site structure of native HtSH and variants in which a glutamate residue in the active-site cavity was replaced by glutamine, alanine, and aspartate. Our biochem., spectroscopic, and theor. studies reveal that at least two active-site states of oxidized HtSH feature an unusual architecture in which the glutamate acts as a terminal ligand of the active-site nickel. This observation demonstrates that crystallog. obsd. glutamate coordination represents a native feature of the enzyme. One of these states is diamagnetic and characterized by a very high stretching frequency of an iron-bound active-site CO ligand. Supported by d.-functional-theory calcns., we identify this state as a high-valent species with a biol. unprecedented formal Ni(IV) ground state. Detailed insights into its structure and dynamics were obtained by ultrafast and two-dimensional IR spectroscopy, demonstrating that it represents a conformationally strained state with unusual bond properties. Our data further show that this state is selectively and reversibly formed under oxic conditions, esp. upon rapid exposure to high O2 levels. We conclude that the kinetically controlled formation of this six-coordinate high-valent state represents a specific and precisely orchestrated stereoelectronic response toward O2 that could protect the enzyme from oxidative damage.
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    Preissler, J.; Reeve, H. A.; Zhu, T.; Nicholson, J.; Urata, K.; Lauterbach, L.; Wong, L. L.; Vincent, K. A.; Lenz, O. Dihydrogen-Driven NADPH Recycling in Imine Reduction and P450-Catalyzed Oxidations Mediated by an Engineered O2-Tolerant Hydrogenase. ChemCatChem. 2020, 12 (19), 4853– 4861,  DOI: 10.1002/cctc.202000763
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    Dihydrogen-Driven NADPH Recycling in Imine Reduction and P450-Catalyzed Oxidations Mediated by an Engineered O2-Tolerant Hydrogenase
    Preissler, Janina; Reeve, Holly A.; Zhu, Tianze; Nicholson, Jake; Urata, Kouji; Lauterbach, Lars; Wong, Luet L.; Vincent, Kylie A.; Lenz, Oliver
    ChemCatChem (2020), 12 (19), 4853-4861CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The O2-tolerant NAD+-reducing hydrogenase (SH) from Ralstonia eutropha (Cupriavidus necator) has already been applied in vitro and in vivo for H2-driven NADH recycling in coupled enzymic reactions with various NADH-dependent oxidoreductases. To expand the scope for application in NADPH-dependent biocatalysis, we introduced changes in the NAD+-binding pocket of the enzyme by rational mutagenesis, and generated a variant with significantly higher affinity for NADP+ than for the natural substrate NAD+, while retaining native O2-tolerance. The applicability of the SH variant in H2-driven NADPH supply was demonstrated by the full conversion of 2-methyl-1-pyrroline into a single enantiomer of 2-methylpyrrolidine catalyzed by a stereoselective imine reductase. In an even more challenging reaction, the SH supported a cytochrome P 450 monooxygenase for the oxidn. of octane under safe H2/O2 mixts. Thus, the re-designed SH represents a versatile platform for atom-efficient, H2-driven cofactor recycling in biotransformations involving NADPH-dependent oxidoreductases.
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    Cammack, R.; Fernandez, V. M.; Hatchikian, E. C. [5] Nickel-Iron Hydrogenase. Methods Enzym. 1994, 243 (1989), 43– 68,  DOI: 10.1016/0076-6879(94)43007-1
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    Yagi, T.; Honya, M.; Tamiya, N. Purification and Properties of Hydrogenases of Different Origins. Biochim. Biophys. Acta (BBA)-Bioenergetics 1968, 153 (3), 699– 705,  DOI: 10.1016/0005-2728(68)90197-7
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    Spencer, P.; Bown, K. J.; Scawen, M. D.; Atkinson, T.; Gore, M. G. Isolation and Characterisation of the Glycerol Dehydrogenase from Bacillus Stearothermophilus. Biochim. Biophys. Acta (BBA)/Protein Struct. Mol. 1989, 994 (3), 270– 279,  DOI: 10.1016/0167-4838(89)90304-X
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    Iyer, P. V.; Ananthanarayan, L. Enzyme Stability and Stabilization-Aqueous and Non-Aqueous Environment. Process Biochem. 2008, 43 (10), 1019– 1032,  DOI: 10.1016/j.procbio.2008.06.004
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    Enzyme stability and stabilization-Aqueous and non-aqueous environment
    Iyer, Padma V.; Ananthanarayan, Laxmi
    Process Biochemistry (Amsterdam, Netherlands) (2008), 43 (10), 1019-1032CODEN: PBCHE5; ISSN:1359-5113. (Elsevier B.V.)
    A review. Enzyme stabilization has notable importance due to increasing no. of enzyme applications. Stabilization of enzymes in order to realize their full potential as catalysts is discussed in the present review. An overview of the denaturation mechanisms in aq. and non-aq. environment is given. Further various methods of enzyme stabilization with respect to their use in aq. and non-aq. environment have been given. Using thermophilic enzymes as the ref. point, a review of stabilization using various approaches like protein engineering, chem. modifications of enzymes and immobilization has been attempted. Finally, it has been stressed that, for selection of a suitable stabilization approach the intended use and possible interactions between the stabilizer-enzyme have to be taken into consideration.
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    Łapińska, U.; Kahveci, Z.; Irwin, N. A. T.; Milner, D. S.; Santoro, A. E.; Richards, T. A.; Pagliara, S. Membrane Permeability Differentiation at the Lipid Divide. PLoS biology 2023, 21 (4), e3002048,  DOI: 10.1371/journal.pbio.3002048
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    Rigoulet, M.; Bouchez, C. L.; Paumard, P.; Ransac, S.; Cuvellier, S.; Duvezin-Caubet, S.; Mazat, J. P.; Devin, A. Cell Energy Metabolism: An Update. Biochim. Biophys. Acta - Bioenerg. 2020, 1861 (11), 148276,  DOI: 10.1016/j.bbabio.2020.148276
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    Cell energy metabolism: An update
    Rigoulet, M.; Bouchez, C. L.; Paumard, P.; Ransac, S.; Cuvellier, S.; Duvezin-Caubet, S.; Mazat, J. P.; Devin, A.
    Biochimica et Biophysica Acta, Bioenergetics (2020), 1861 (11), 148276CODEN: BBBEB4; ISSN:0005-2728. (Elsevier B.V.)
    A review. In living cells, growth is the result of coupling between substrate catabolism and multiple metabolic processes that take place during net biomass formation and maintenance processes. During growth, both ATP/ADP and NADH/NAD+ mols. play a key role. Cell energy metab. hence refers to metabolic pathways involved in ATP synthesis linked to NADH turnover. Two main pathways are thus involved in cell energy metab.: glycolysis/fermn. and oxidative phosphorylation. Glycolysis and mitochondrial oxidative phosphorylation are intertwined through thermodn. and kinetic constraints that are reviewed herein. Further, our current knowledge of short-term and long term regulation of cell energy metab. will be reviewed using examples such as the Crabtree and the Warburg effect.
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    Pollak, N.; Dölle, C.; Ziegler, M. The Power to Reduce: Pyridine Nucleotides - Small Molecules with a Multitude of Functions. Biochem. J. 2007, 402 (2), 205– 218,  DOI: 10.1042/BJ20061638
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    The power to reduce: pyridine nucleotides - small molecules with a multitude of functions
    Pollak, Nadine; Doelle, Christian; Ziegler, Mathias
    Biochemical Journal (2007), 402 (2), 205-218CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
    A review. The pyridine nucleotides NAD and NADP play vital roles in metabolic conversions as signal transducers and in cellular defense systems. Both coenzymes participate as electron carriers in energy transduction and biosynthetic processes. Their oxidized forms, NAD+ and NADP+, have been identified as important elements of regulatory pathways. In particular, NAD+ serves as a substrate for ADP-ribosylation reactions and for the Sir2 family of NAD+-dependent protein deacetylases as well as a precursor of the calcium mobilizing mol. cADPr (cyclic ADP-ribose). The conversions of NADP+ into the 2'-phosphorylated form of cADPr or to its nicotinic acid deriv., NAADP, also result in the formation of potent intracellular calcium-signalling agents. Perhaps, the most crit. function of NADP is in the maintenance of a pool of reducing equiv. which is essential to counteract oxidative damage and for other detoxifying reactions. It is well known that the NADPH/NADP+ ratio is usually kept high, in favor of the reduced form. Research within the past few years has revealed important insights into how the NADPH pool is generated and maintained in different subcellular compartments. Moreover, tremendous progress in the mol. characterization of NAD kinases has established these enzymes as vital factors for cell survival. In the present review, we summarize recent advances in the understanding of the biosynthesis and signalling functions of NAD(P) and highlight the new insights into the mol. mechanisms of NADPH generation and their roles in cell physiol.
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    Tishkov, V. I.; Popov, V. O. Protein Engineering of Formate Dehydrogenase. Biomol. Eng. 2006, 23 (2–3), 89– 110,  DOI: 10.1016/j.bioeng.2006.02.003
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    Protein engineering of formate dehydrogenase
    Tishkov, Vladimir I.; Popov, Vladimir O.
    Biomolecular Engineering (2006), 23 (2-3), 89-110CODEN: BIENFV; ISSN:1389-0344. (Elsevier B.V.)
    A review. NAD-dependent formate dehydrogenase (FDH; EC 1.2.1.2) is one of the best enzymes for the purpose of NADH regeneration in dehydrogenase-based synthesis of optically active compds. Low operational stability and high prodn. cost of native FDHs limit their application in com. prodn. of chiral compds. Here, the authors summarize the results on engineering of bacterial and yeast FDHs aimed at improving their chem. and thermal stability, catalytic activity, switch in coenzyme specificity from NAD to NADP, and overexpression in Escherichia coli cells.
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    Bouzon, M.; Döring, V.; Dubois, I.; Berger, A.; Stoffel, G. M. M.; Ramirez, L. C.; Meyer, S. N.; Fouré, M.; Roche, D.; Perret, A.; Erb, T. J.; Bar-Even, A.; Lindner, S. N. Change in Cofactor Specificity of Oxidoreductases by Adaptive Evolution of an Escherichia coli Nadph-Auxotrophic Strain. MBio 2021, 12 (4), e00329-21,  DOI: 10.1128/mBio.00329-21
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    Chánique, A. M.; Parra, L. P. Protein Engineering for Nicotinamide Coenzyme Specificity in Oxidoreductases: Attempts and Challenges. Front. Microbiol. 2018,  DOI: 10.3389/fmicb.2018.00194
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    Rydström, J.; Hoek, J. B.; Ernster, L. 2 Nicotinamide Nucleotide Transhydrogenases. The Enzymes 1976, 13, 51– 88,  DOI: 10.1016/S1874-6047(08)60240-1
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    Physiological roles of nicotinamide nucleotide transhydrogenase
    Hoek, Jan B.; Rydstroem, Jan
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    A review, with 93 refs., on the kinetic and thermodn. characteristics of the title transhydrogenase and its functions, e.g. as a redox buffer in the supply of reducing equiv., and protection of the mitochondria NADP redox state, as well as hormone effects on the enzyme.
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    Pedersen, A.; Karlsson, G. B.; Rydström, J. Proton-Translocating Transhydrogenase: An Update of Unsolved and Controversial Issues. J. Bioenerg. Biomembr. 2008, 40 (5), 463– 473,  DOI: 10.1007/s10863-008-9170-x
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    Proton-translocating transhydrogenase: an update of unsolved and controversial issues
    Pedersen, Anders; Karlsson, Goeran B.; Rydstroem, Jan
    Journal of Bioenergetics and Biomembranes (2008), 40 (5), 463-473CODEN: JBBID4; ISSN:0145-479X. (Springer)
    A review. H+-translocating transhydrogenases, reducing NADP to NADH through hydride transfer, are membrane enzymes utilizing the electrochem. proton gradient for NADPH generation. These enzymes have important physiol. roles in the maintenance of e.g., reduced glutathione, relevant for essentially all cell types. Following x-ray crystallog. and structural resoln. of the sol. substrate-binding domains, mechanistic aspects of the hydride transfer reaction are beginning to be resolved. However, the structure of the intact enzyme is still unknown. Key questions regarding the coupling mechanism, i.e., the mechanism of proton translocation, are addressed using the sep. expressed substrate-binding domains. Important aspects are therefore which functions and properties of mainly the sol. NADP(H)-binding domain (but also the NAD(H)-binding domain) are relevant for proton translocation, how the sol. domains communicate with the membrane domain, and the mechanism of proton translocation through the membrane domain.
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    Leung, J. H.; Schurig-Briccio, L. A.; Yamaguchi, M.; Moeller, A.; Speir, J. A.; Gennis, R. B.; Stout, C. D. Division of Labor in Transhydrogenase by Alternating Proton Translocation and Hydride Transfer. Science. 2015, 347 (6218), 178– 181,  DOI: 10.1126/science.1260451
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    Division of labor in transhydrogenase by alternating proton translocation and hydride transfer
    Leung, Josephine H.; Schurig-Briccio, Lici A.; Yamaguchi, Mutsuo; Moeller, Arne; Speir, Jeffrey A.; Gennis, Robert B.; Stout, Charles D.
    Science (Washington, DC, United States) (2015), 347 (6218), 178-181CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    NADPH/NADP+ (the reduced form of NADP+/NADP) homeostasis is crit. for countering oxidative stress in cells. Nicotinamide nucleotide transhydrogenase (TH), a membrane enzyme present in both bacteria and mitochondria, couples the proton motive force (PMF) to the generation of NADPH. We present the 2.8 Å crystal structure of the transmembrane proton channel domain of TH from Thermus thermophilus and the 6.9 Å crystal structure of the entire enzyme (holo-TH). The membrane domain crystd. as a sym. dimer, with each protomer contg. a putative proton channel. The holo-TH is a highly asym. dimer with the NADP(H)-binding domain (dIII) in two different orientations. This unusual arrangement suggests a catalytic mechanism in which the two copies of dIII alternatively function in proton translocation and hydride transfer.
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    Kampjut, D.; Sazanov, L. A. Structure and Mechanism of Mitochondrial Proton-Translocating Transhydrogenase. Nature 2019, 573 (7773), 291– 295,  DOI: 10.1038/s41586-019-1519-2
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    129
    Structure and mechanism of mitochondrial proton-translocating transhydrogenase
    Kampjut, Domen; Sazanov, Leonid A.
    Nature (London, United Kingdom) (2019), 573 (7773), 291-295CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    Proton-translocating transhydrogenase (also known as nicotinamide nucleotide transhydrogenase (NNT)) is found in the plasma membranes of bacteria and the inner mitochondrial membranes of eukaryotes. NNT catalyzes the transfer of a hydride between NADH and NADP+, coupled to the translocation of one proton across the membrane. Its main physiol. function is the generation of NADPH, which is a substrate in anabolic reactions and a regulator of oxidative status; however, NNT may also fine-tune the Krebs cycle1,2. NNT deficiency causes familial glucocorticoid deficiency in humans and metabolic abnormalities in mice, similar to those obsd. in type II diabetes3,4. The catalytic mechanism of NNT has been proposed to involve a rotation of around 180° of the entire NADP(H)-binding domain that alternately participates in hydride transfer and proton-channel gating. However, owing to the lack of high-resoln. structures of intact NNT, the details of this process remain unclear5,6. Here we present the cryo-electron microscopy structure of intact mammalian NNT in different conformational states. We show how the NADP(H)-binding domain opens the proton channel to the opposite sides of the membrane, and we provide structures of these two states. We also describe the catalytically important interfaces and linkers between the membrane and the sol. domains and their roles in nucleotide exchange. These structures enable us to propose a revised mechanism for a coupling process in NNT that is consistent with a large body of previous biochem. work. Our results are relevant to the development of currently unavailable NNT inhibitors, which may have therapeutic potential in ischemia reperfusion injury, metabolic syndrome and some cancers7-9.
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    Enander, K.; Rydström, J. Energy-Linked Nicotinamide Nucleotide Transhydrogenase. Kinetics and Regulation of Purified and Reconstituted Transhydrogenase from Beef Heart Mitochondria. J. Biol. Chem. 1982, 257 (24), 14760– 14766,  DOI: 10.1016/S0021-9258(18)33345-3
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    130
    Energy-linked nicotinamide nucleotide transhydrogenase. Kinetics and regulation of purified and reconstituted transhydrogenase from beef heart mitochondria
    Enander, Kirsten; Rydstroem, Jan
    Journal of Biological Chemistry (1982), 257 (24), 14760-6CODEN: JBCHA3; ISSN:0021-9258.
    Purified and reconstituted nicotinamide nucleotide transhydrogenase (I) from bovine heart mitochondria was investigated with respect to kinetic and regulatory properties in uncoupled and coupled liposomes. Double reciprocal plots of initial velocities for the redn. of NADPH vs. substrate concns. were convergent and intersecting on or close to the abscissa, indicating a ternary complex mechanism. The effect of site-specific inhibitors indicated that the order of addn. of the substrates to I was random. Reconstituted I uncoupled by FCCP revealed kinetic properties that were indicative of energization, i.e., an increased and decreased affinity for NADP and NAD, resp., suggesting that reconstituted I is maintained in an activated conformation. An increased extent of coupling caused a progressively increasing change in the same direction. Apparently, the uncoupler-dependent enhancement of the rate of redn. of NAD by NADPH is due to a decreased Km for NAD. Reconstituted I catalyzed a transhydrogenation between NADH and 3-acetylpyridine adenine dinucleotide (oxidized) in the presence of NADPH. Reconstituted I also catalyzed the redn. of thio-NADP by NADPH in the presence of NADH. Both reactions occurred indirectly through the generation of NADP and NAD, resp., and not directly through a reduced I intermediate. A H+-pump mechanism is proposed for I which involves a dimeric form of I where the 2 subunits alternate in H+ pumping.
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    Graf, S. S.; Hong, S.; Müller, P.; Gennis, R.; von Ballmoos, C. Energy Transfer between the Nicotinamide Nucleotide Transhydrogenase and ATP Synthase of Escherichia coli. Sci. Rep. 2021, 11 (1), 1– 12,  DOI: 10.1038/s41598-021-00651-6
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    Sauer, U.; Canonaco, F.; Heri, S.; Perrenoud, A.; Fischer, E. The Soluble and Membrane-Bound Transhydrogenases UdhA and PntAB Have Divergent Functions in NADPH Metabolism of Escherichia coli. J. Biol. Chem. 2004, 279 (8), 6613– 6619,  DOI: 10.1074/jbc.M311657200
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    132
    The soluble and membrane-bound transhydrogenases UdhA and PntAB have divergent functions in NADPH metabolism of Escherichia coli
    Sauer, Uwe; Canonaco, Fabrizio; Heri, Sylvia; Perrenoud, Annik; Fischer, Eliane
    Journal of Biological Chemistry (2004), 279 (8), 6613-6619CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Pentose phosphate pathway and isocitrate dehydrogenase are generally considered to be the major sources of the anabolic reductant NADPH. As one of very few microbes, Escherichia coli contains two transhydrogenase isoforms with unknown physiol. function that could potentially transfer electrons directly from NADH to NADP+ and vice versa. Using defined mutants and metabolic flux anal., we identified the proton-translocating transhydrogenase PntAB as a major source of NADPH in E. coli. During std. aerobic batch growth on glucose, 35-45% of the NADPH that is required for biosynthesis was produced via PntAB, whereas pentose phosphate pathway and isocitrate dehydrogenase contributed 35-45% and 20-25%, resp. The energy-independent transhydrogenase UdhA, in contrast, was essential for growth under metabolic conditions with excess NADPH formation, i.e. growth on acetate or in a phosphoglucose isomerase mutant that catabolized glucose through the pentose phosphate pathway. Thus, both isoforms have divergent physiol. functions: energy-dependent redn. of NADP+ with NADH by PntAB and reoxidn. of NADPH by UdhA. Expression appeared to be modulated by the redox state of cellular metab., because genetic and environmental manipulations that increased or decreased NADPH formation down-regulated pntA or udhA transcription, resp. The two transhydrogenase isoforms provide E. coli primary metab. with an extraordinary flexibility to cope with varying catabolic and anabolic demands, which raises two general questions: why do only a few bacteria contain both isoforms, and how do other organisms manage NADPH metab.
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    Boonstra, B.; Bjorklund, L.; French, C. E.; Wainwright, I.; Bruce, N. C. Cloning of the Sth Gene from Azotobacter Vinelandii and Construction of Chimeric Soluble Pyridine Nucleotide Transhydrogenases. FEMS Microbiol. Lett. 2000, 191 (1), 87– 93,  DOI: 10.1111/j.1574-6968.2000.tb09323.x
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    Cloning of the sth gene from Azotobacter vinelandii and construction of chimeric soluble pyridine nucleotide transhydrogenases
    Boonstra, B.; Bjorklund, L.; French, C. E.; Wainwright, I.; Bruce, N. C.
    FEMS Microbiology Letters (2000), 191 (1), 87-93CODEN: FMLED7; ISSN:0378-1097. (Elsevier Science B.V.)
    The gene encoding the sol. pyridine nucleotide transhydrogenase (STH) of Azotobacter vinelandii was cloned and sequenced. This is the third sth gene identified and further defines a new subfamily within the flavoprotein disulfide oxidoreductases. The three STHs identified all lack one of the redox active cysteines that are characteristic for this large family of enzymes, and instead they contain a conserved threonine residue at this position. The recombinant A. vinelandii enzyme was purified to homogeneity and shown to form filamentous structures different from those of Pseudomonas fluorescens and Escherichia coli STH. Chimeric STHs were constructed which showed that the C-terminal region is important for polymer formation. The A. vinelandii STH contg. the C-terminal region of P. fluorescens or E. coli STH showed structures resembling those of the STH contributing the C-terminal portion of the protein.
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    Voordouw, G.; Veeger, C.; Breemen, J. F. L.; Bruggen, E. F. J. Structure Of Pyridine Nucleotide Transhydrogenase From Azotobacter Vinelandii. Eur. J. Biochem. 1979, 98 (2), 447– 454,  DOI: 10.1111/j.1432-1033.1979.tb13205.x
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    Structure of pyridine nucleotide transhydrogenase from azotobacter vinelandii
    Voordouw, Gerrit; Veeger, Cees; Van Breemen, Jan F. L.; Van Bruggen, Ernst F. J.
    European Journal of Biochemistry (1979), 98 (2), 447-54CODEN: EJBCAI; ISSN:0014-2956.
    Pyridine nucleotide transhydrogenase (I) of A. vinelandii purified by affinity chromatog. consisted of a mixt. of polydisperse rods at neutral pH. No other structures were seen by electron microscopy. A high pH (8.5-9.0) the rods depolymd. Complete depolymn. was achieved in 0.1M Tris-Cl pH 9.0. Depolymd. I has a mol. wt. of 421,000 (sedimentation equil.), sedimentation coeff. of 15 S, and its Stokes' radius is 7 nm. Since gel electrophoresis in the presence of Na dodecyl sulfate show that I consists of a single polypeptide chain of mol. wt. 54 × 103, it follows that depolymd. I has an octameric quaternary structure. This octamer may serve as the functional monomeric unit (unimer) from which the polymeric form of I is constructed. Gel filtration and sucrose gradient centrifugation studies of cell-free exts. show the unimer to be the predominant active species.
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    Partipilo, M.; Yang, G.; Mascotti, M. L.; Wijma, H. J.; Slotboom, D. J.; Fraaije, M. W. A Conserved Sequence Motif in the Escherichia coli Soluble FAD-Containing Pyridine Nucleotide Transhydrogenase Is Important for Reaction Efficiency. J. Biol. Chem. 2022, 298 (9), 102304,  DOI: 10.1016/j.jbc.2022.102304
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    A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency
    Partipilo, Michele; Yang, Guang; Mascotti, Maria Laura; Wijma, Hein J.; Slotboom, Dirk Jan; Fraaije, Marco W.
    Journal of Biological Chemistry (2022), 298 (9), 102304CODEN: JBCHA3; ISSN:1083-351X. (Elsevier Inc.)
    Sol. pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equiv. between the two nicotinamide cofactors. The sol. transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equiv. toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochem. characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with mol. oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational anal. revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with mol. oxygen and the importance of the evolutionarily conserved sequence motif.
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    Cao, Z.; Song, P.; Xu, Q.; Su, R.; Zhu, G. Overexpression and Biochemical Characterization of Soluble Pyridine Nucleotide Transhydrogenase from Escherichia coli. FEMS Microbiol. Lett. 2011, 320 (1), 9– 14,  DOI: 10.1111/j.1574-6968.2011.02287.x
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    Overexpression and biochemical characterization of soluble pyridine nucleotide transhydrogenase from Escherichia coli
    Cao, Zhengyu; Song, Ping; Xu, Qin; Su, Ruirui; Zhu, Guoping
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    Sol. pyridine nucleotide transhydrogenase (STH) is an energy-independent flavoprotein that directly catalyzes hydride transfer between NAD(H) and NADP(H) to maintain homeostasis of these 2 redox cofactors. Here, the sth gene of E. coli was cloned and expressed as a fused protein (EcSTH). Purified EcSTH displayed maximal activity at 35° and pH 7.5. Heat-inactivation studies showed that EcSTH retained 50% activity after 5 h at 50°. The enzyme was stable at 4° for 25 days. The apparent Km values of EcSTH were 68.29 μM for NADPH and 133.2 μM for thio-NAD. The kcat/Km ratios showed that EcSTH had a 1.25-fold preference for NADPH over thio-NAD. Product inhibition studies showed that EcSTH activity was strongly inhibited by excess NADPH, but not by thio-NAD. EcSTH activity was enhanced by 2 mM adenine nucleotide and inhibited by divalent metal ions, including Mn2+, Co2+, Zn2+, Ni2+, and Cu2+. However, after preincubation for 30 min, most divalent metal ions had little effect on EcSTH activity, except Zn2+, Ni2+, and Cu2+. This enzymic anal. provides important basic knowledge for EcSTH utilization.
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    Ighodaro, O. M.; Akinloye, O. A. First Line Defence Antioxidants-Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione Peroxidase (GPX): Their Fundamental Role in the Entire Antioxidant Defence Grid. Alexandria J. Med. 2018, 54 (4), 287– 293,  DOI: 10.1016/j.ajme.2017.09.001
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  • Abstract

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    Figure 1

    Figure 1. NAD(H) and NADP(H) occurrence among the enzymatic reactions of Escherichia coli. Metabolites involved in cellular reactions tend to follow a power-law distribution (generically described by the equation y = C/xa), in which the majority of the metabolites is involved in a small number of reactions while a small group of metabolites takes part in up to 100 enzyme-mediated reactions. On the left y-axis is reported the absolute number of metabolites (n), and on the right y-axis is shown the same number of metabolites in terms of percentage (%). The diagonal approximates the linearity of the power-law distribution in a double logarithmic scale graph, as also shown by Schmidt et al. (17) The restricted group of metabolites involved in more than a hundred reactions represent exceptions to the power-law distribution, and are called hub metabolites. The upper right inset highlights the number of reactions dependent on NAD+ (red diamond), NADH (red-black diamond), NADP+ (blue diamond), and NADPH (blue-black diamond). The data set was extracted from the Escherichia coli Metabolome Database (http://www.ecmdb.ca).

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    Figure 2

    Figure 2. Overview of the standard reduction potentials (E°′, at 25 °C, pH 7.0) of the half-reactions of candidate electron donors. (32−36)

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    Figure 3

    Figure 3. Strategies for the orthogonal provision of reducing power in synthetic cells. Both the electron donors for the nicotinamide coenzymes and the respective reaction products have two possible ways to move across the membrane: by protein-mediated action (A) or by unassisted permeation (B–D). (A) Phosphite is internalized by an ATP-binding cassette (ABC) transporter (shown in green), while its reaction product phosphate can be exported in symport with a proton (in yellow) or a sodium ion (in orange), or by facilitated-diffusion (in red). (B–D) Formate, molecular hydrogen and glycerol, as well as carbon dioxide and dihydroxyacetone freely and readily permeate the phospholipid bilayer. In order to transfer the hydrides from the donor to the NAD(P)+ cofactors, the cell-like compartments need to be equipped with specific dehydrogenase(s).

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    Figure 4

    Figure 4. Phosphite dehydrogenase from P. stutzeri WM 88. (A) The homodimeric assembly of the holoenzyme in complex with NAD+ and sulfite, specific inhibitor of PDH (PDB entry: 4E5K). (B) A zoom into the PDH active site. The conserved catalytic triad Arg237, Glu266, and His292 allows catalysis with a proposed acid–base mechanism. The nucleophilic attack on the phosphite (here replaced by the competitive inhibitor sulfite) is initiated by histidine─via a water molecule─and is oriented by the combined action of arginine and glutamate, resulting in the reduction of the nicotinamide coenzyme.

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    Figure 5

    Figure 5. Formate dehydrogenase from Pseudomonas sp. 101. (A) The nonmetal containing FDH is homodimeric, here shown in complex with NAD+ and azide (N3), an inhibitor analogue of formate (PDB entry: 2NAD). (B) The catalysis mediated by FDH requires the involvement of multiple amino acids. His332 and Ser334 establish hydrogen bonds with the carboxamide group of the nicotinamide ring. Through a hydrogen bond with His332, Gln313 plays a key role in the FDH reaction, ensuring its characteristic wide pH optimum. (102) The binding with formate (here replaced with the analogue, azide) is established through electrostatic interactions with the positively charged substituents of Arg284 and Asn146, while Ile122 favors the charge counterbalance of the substrate. The hydrophobic cluster (Phe97 and Pro98) promotes the spatial constriction and consequent destabilization of formate into the configuration of the reaction intermediate. Distances and other amino acids involved to a minor extent in the catalytic mechanism are here omitted, but discussed in detail somewhere else. (73,103)

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    Figure 6

    Figure 6. Soluble [NiFe]-hydrogenase structure. (A) The scheme of the subunit and cofactor assembly. The hydrogenase module HoxHY initiates the dihydrogen oxidation (HoxH in blue, HoxY in cyan). Different iron–sulfur clusters (red and yellow balls) allow the electron transfer from the hydrogenase to the diaphorase subunit HoxFU (HoxU in gray, HoxF in green), leading to the NAD+ reduction into NADH. The binding of 2 identical HoxI subunits (in yellow) to HoxF generates an hexameric assembly that promotes the reductive-protein activation by NADPH. (B) The tetrameric structure of SH from Hydrogenophilus thermoluteolus TH-1 (PDB entry: 5XF9). The color scheme is the same used in A.

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    Figure 7

    Figure 7. Glycerol dehydrogenase from Geobacillus stearothermophilus. (A) The enzyme has an homo-octameric assembly in solution, here illustrated in complex with NAD+ (PDB entry: 1JQ5). (B) Overview of the active site of GlyDH in complex with the substrates. By superimposing the structures in complex with NAD+ and glycerol (PDB entries: 1JQ5, 1JQA), the essential amino acids to accommodate both substrates are highlighted. Asp100, Ser127, and Leu129 establish a hydrogen bond network with the carboxamide group of the nicotinamide moiety. Glycerol is correctly oriented toward the catalytic center through van der Waals interactions between Phe247 and the carbon atoms of the substrate. The coordination of zinc is mediated by dipole interactions with Asp173, His256, His274, and a water molecule.

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    Figure 8

    Figure 8. Transhydrogenases in synthetic cells. (A) Scheme of the redox regeneration. Following the luminal access of the electron donor (passively indicated by the dashed arrow, actively by the solid arrow), NAD+ (left side) or NADP+ (right side) is reduced by an upstream specific dehydrogenase. A downstream transhydrogenase transfers electrons from one cofactor to another, overcoming the limitation of cofactor specificity, and reoxidizing the cosubstrate NAD(P)H during transhydrogenation. An additional sink enzymatic reaction can take advantage of the new reduced cofactor, while pulling the transhydrogenase reaction out of equilibrium. (B) Structural organization of membrane transhydrogenases. On top, the scheme of the protein assembly. All known mTHs are organized into three distinct domains: dI, NAD(H)-binding in yellow; dII, transmembrane in cyan; dIII, NADP(H)-binding in red. On bottom, the heteromeric structure of the holo-enzyme NNT from Ovis aries mitochondria (PDB entry: 6QTI) in complex with NAD+ and NADP+. (C) Structural and catalytic insight of soluble transhydrogenases. On the left, the monomeric predicted structure of the soluble transhydrogenase from E. coli K-12 (AlphaFold entry: AF-P27306) embedding the prosthetic group─FAD─upon superimposition with the related lipoamide dehydrogenase from Thermus thermophilus HB8 (PDB entry: 2EQ7). On the right is a zoom into the FAD-binding domain in complex with NAD+, highlighting cysteine 45 and threonine 50, as they are conserved amino acids in sTHs for efficient catalytic activity.

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  • References

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    This article references 137 other publications.

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      Micelles that can catalyze their replication have been described recently. In the previous expts., micelles (or bilayer vesicles) were always present in the initial reaction mixt., i.e., the system was presented with the bounded structures required for autocatalysis. Here a system is described in which autocatalytic micelles are formed from amphiphiles that are themselves generated from a hydrolysis reaction in the absence of compartmental structures. Alk. hydrolysis of Et caprylate (itself insol. in water) yields sodium caprylate, initially at a very slow rate; but as soon as sufficient caprylate is formed for aggregation into micelles to take place, there is an exponential increase in reaction rate owing to micellar catalysis. These self-assembling surfactant structures may consequently provide a model system for studies of prebiotic chem. The possible relevance of this process to prebiotic chem. is emphasized by the observation that the micelles can be converted into more-robust vesicles by a pH change induced by dissolved CO2.
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      Autopoietic Self-Reproduction of Fatty Acid Vesicles
      Walde, Peter; Wick, Roger; Fresta, Massimo; Mangone, Annarosa; Luisi, Pier Luigi
      Journal of the American Chemical Society (1994), 116 (26), 11649-54CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Conditions are described under which vesicles formed by caprylic acid and oleic acid in H2O are able to undergo autopoietic self-reprodn.-e.g. an increase of their population no. due to a reaction which takes place within the spherical boundary of the vesicles themselves. This is achieved by letting a certain amt. of the neat H2O-insol. caprylic or oleic anhydride hydrolyze at alk. pH: the initial increase of the concn. of the released acid/carboxylate is extremely slow (several days to reach the conditions for spontaneous vesicle formation), but afterwards, the presence of vesicles brings about a rapid 2nd phase leading to more and more vesicles being formed in an overall autocatalytic process. The catalytic power of the caprylic acid and oleic acid vesicles toward the hydrolysis of the corresponding anhydride is documented in a set of independent expts. In these expts., the hydrolysis was carried out in the presence of vesicles at a pH corresponding approx. to the pK of the acid in the vesicles. The process of autopoietic self-reprodn. of caprylic acid and oleic acid vesicles is studied as a function of temp.: by increasing temp. (≤ 70°), the exponential time progress of vesicle formation tends to become steeper while the long initial slow phase is significantly shortened. The caprylic acid and oleic acid vesicles are characterized by electron microscopy and by detg. their internal vol. The question whether and to what extent these vesicles form a classic chem. equil. system (e.g., in which the free surfactant is in equil. with the aggregates) is also studied.
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    3. 3
      Noireaux, V.; Libchaber, A. A Vesicle Bioreactor as a Step toward an Artificial Cell Assembly. Proc. Natl. Acad. Sci. U. S. A. 2004, 101 (51), 17669– 17674,  DOI: 10.1073/pnas.0408236101
      3
      A vesicle bioreactor as a step toward an artificial cell assembly
      Noireaux, Vincent; Libchaber, Albert
      Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (51), 17669-17674CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      An Escherichia coli cell-free expression system is encapsulated in a phospholipid vesicle to build a cell-like bioreactor. Large unilamellar vesicles contg. exts. are produced in an oil-ext. emulsion. To form a bilayer the vesicles are transferred into a feeding soln. that contains ribonucleotides and amino acids. Transcription-translation of plasmid genes is isolated in the vesicles. Whereas in bulk soln. expression of enhanced GFP (eGFP) stops after 2 h, inside the vesicle permeability of the membrane to the feeding soln. prolongs the expression for up to 5 h. To solve the energy and material limitations and increase the capacity of the reactor, the α-hemolysin pore protein from Staphylococcus aureus is expressed inside the vesicle to create a selective permeability for nutrients. The reactor can then sustain expression for up to 4 days with a protein prodn. of 30 μM after 4 days. Oxygen diffusion and osmotic pressure are crit. parameters to maintain expression and avoid vesicle burst.
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    4. 4
      Kita, H.; Matsuura, T.; Sunami, T.; Hosoda, K.; Ichihashi, N.; Tsukada, K.; Urabe, I.; Yomo, T. Replication of Genetic Information with Self-Encoded Replicase in Liposomes. ChemBioChem. 2008, 9 (15), 2403– 2410,  DOI: 10.1002/cbic.200800360
      4
      Replication of genetic information with self-encoded replicase in liposomes
      Kita, Hiroshi; Matsuura, Tomoaki; Sunami, Takeshi; Hosoda, Kazufumi; Ichihashi, Norikazu; Tsukada, Koji; Urabe, Itaru; Yomo, Tetsuya
      ChemBioChem (2008), 9 (15), 2403-2410CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      In all living systems, the genome is replicated by proteins that are encoded within the genome itself. This universal reaction is essential to allow the system to evolve. Here, we have constructed a simplified system involving encapsulated macromols. termed a "self-encoding system", in which the genetic information is replicated by self-encoded replicase in liposomes. That is, the universal reaction was reconstituted within a microcompartment bound by a lipid bilayer. The system was assembled by using one template RNA sequence as the information mol. and on in vitro translation system reconstituted from purified translation factors as the machinery for decoding the information. In this system, the catalytic subunit of Qβ replicase is synthesized from the template RNA that encodes the protein. The replicase then replicates the template RNA that was used for its prodn. This in-liposome self-encoding system is one of the simplest such systems available; it consists of only 144 gene products, while the information and the function for its replication are encoded on different mols. and are compartmentalized into the microenvironment for evolvability.
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    5. 5
      Luisi, P. L.; Ferri, F.; Stano, P. Approaches to Semi-Synthetic Minimal Cells: A Review. Naturwissenschaften 2006, 93 (1), 1– 13,  DOI: 10.1007/s00114-005-0056-z
      5
      Approaches to semi-synthetic minimal cells: a review
      Luisi, Pier Luigi; Ferri, Francesca; Stano, Pasquale
      Naturwissenschaften (2006), 93 (1), 1-13CODEN: NATWAY; ISSN:0028-1042. (Springer)
      Following is a synthetic review on the minimal living cell, defined as an artificial or a semi-artificial cell having the minimal and sufficient no. of components to be considered alive. We describe concepts and expts. based on these constructions, and we point out that an operational definition of minimal cell does not define a single species, but rather a broad family of interrelated cell-like structures. The relevance of these researches, considering that the minimal cell should also correspond to the early simple cell in the origin of life and early evolution, is also explained. In addn., we present detailed data in relation to minimal genome, with observations cited by several authors who agree on setting the theor. full-fledged minimal genome to a figure between 200 and 300 genes. However, further theor. assumptions may significantly reduce this no. (i.e. by eliminating ribosomal proteins and by limiting DNA and RNA polymerases to only a few, less specific mol. species). Generally, the exptl. approach to minimal cells consists in utilizing liposomes as cell models and in filling them with genes/enzymes corresponding to minimal cellular functions. To date, a few research groups have successfully induced the expression of single proteins, such as the green fluorescence protein, inside liposomes. Here, different approaches are described and compared. Present constructs are still rather far from the minimal cell, and exptl. as well as theor. difficulties opposing further redn. of complexity are discussed. While most of these minimal cell constructions may represent relatively poor imitations of a modern full-fledged cell, further studies will begin precisely from these constructs. In conclusion, we give a brief outline of the next possible steps on the road map to the minimal cell.
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    6. 6
      Forster, A. C.; Church, G. M. Towards Synthesis of a Minimal Cell. Mol. Syst. Biol. 2006, 2 (1), 45,  DOI: 10.1038/msb4100090
      6
      Towards synthesis of a minimal cell
      Forster Anthony C; Church George M
      Molecular systems biology (2006), 2 (), 45 ISSN:.
      Construction of a chemical system capable of replication and evolution, fed only by small molecule nutrients, is now conceivable. This could be achieved by stepwise integration of decades of work on the reconstitution of DNA, RNA and protein syntheses from pure components. Such a minimal cell project would initially define the components sufficient for each subsystem, allow detailed kinetic analyses and lead to improved in vitro methods for synthesis of biopolymers, therapeutics and biosensors. Completion would yield a functionally and structurally understood self-replicating biosystem. Safety concerns for synthetic life will be alleviated by extreme dependence on elaborate laboratory reagents and conditions for viability. Our proposed minimal genome is 113 kbp long and contains 151 genes. We detail building blocks already in place and major hurdles to overcome for completion.
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    7. 7
      Schwille, P. Bottom-Up Synthetic Biology: Engineering in a Tinkerer ’ s World. Science. 2011, 333 (6047), 1252– 1254,  DOI: 10.1126/science.1211701
      7
      Bottom-Up Synthetic Biology: Engineering in a Tinkerer's World
      Schwille, Petra
      Science (Washington, DC, United States) (2011), 333 (6047), 1252-1254CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      A review. How synthetic can "synthetic biol." be A literal interpretation of the name of this new life science discipline invokes expectations of the systematic construction of biol. systems with cells being built module by module-from the bottom up. But can this possibly be achieved, taking into account the enormous complexity and redundancy of living systems, which distinguish them quite remarkably from design features that characterize human inventions There are several recent developments in biol., in tight conjunction with quant. disciplines, that may bring this literal perspective into the realm of the possible. However, such bottom-up engineering requires tools that were originally designed by nature's greatest tinkerer: evolution.
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    8. 8
      Caschera, F.; Noireaux, V. Integration of Biological Parts toward the Synthesis of a Minimal Cell. Curr. Opin. Chem. Biol. 2014, 22, 85– 91,  DOI: 10.1016/j.cbpa.2014.09.028
      8
      Integration of biological parts toward the synthesis of a minimal cell
      Caschera, Filippo; Noireaux, Vincent
      Current Opinion in Chemical Biology (2014), 22 (), 85-91CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
      A review. Various approaches are taken to construct synthetic cells in the lab., a challenging goal that became exptl. imaginable over the past two decades. The construction of protocells, which explores scenarios of the origin of life, has been the original motivations for such projects. With the advent of the synthetic biol. era, bottom-up engineering approaches to synthetic cells are now conceivable. The modular design emerges as the most robust framework to construct a minimal cell from natural mol. components. Although significant advances have been made for each piece making this complex puzzle, the integration of the three fundamental parts, information-metab.-self-organization, into cell-sized liposomes capable of sustained reprodn. has failed so far. Our inability to connect these three elements is also a major limitation in this research area. New methods, such as machine learning coupled to high-throughput techniques, should be exploited to accelerate the cell-free synthesis of complex biochem. systems.
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    9. 9
      Olivi, L.; Berger, M.; Creyghton, R. N. P.; De Franceschi, N.; Dekker, C.; Mulder, B. M.; Claassens, N. J.; ten Wolde, P. R.; van der Oost, J. Towards a Synthetic Cell Cycle. Nat. Commun. 2021, 12 (1), 4531,  DOI: 10.1038/s41467-021-24772-8
      9
      Towards a synthetic cell cycle
      Olivi, Lorenzo; Berger, Mareike; Creyghton, Ramon N. P.; De Franceschi, Nicola; Dekker, Cees; Mulder, Bela M.; Claassens, Nico J.; ten Wolde, Pieter Rein; van der Oost, John
      Nature Communications (2021), 12 (1), 4531CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Abstr.: Recent developments in synthetic biol. may bring the bottom-up generation of a synthetic cell within reach. A key feature of a living synthetic cell is a functional cell cycle, in which DNA replication and segregation as well as cell growth and division are well integrated. Here, we describe different approaches to recreate these processes in a synthetic cell, based on natural systems and/or synthetic alternatives. Although some individual machineries have recently been established, their integration and control in a synthetic cell cycle remain to be addressed. In this Perspective, we discuss potential paths towards an integrated synthetic cell cycle.
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    10. 10
      Pols, T.; Sikkema, H. R.; Gaastra, B. F.; Frallicciardi, J.; Śmigiel, W. M.; Singh, S.; Poolman, B. A Synthetic Metabolic Network for Physicochemical Homeostasis. Nat. Commun. 2019, 10 (1), 1– 13,  DOI: 10.1038/s41467-019-12287-2
      10
      A synthetic metabolic network for physicochemical homeostasis
      Pols, Tjeerd; Sikkema, Hendrik R.; Gaastra, Bauke F.; Frallicciardi, Jacopo; Smigiel, Wojciech M.; Singh, Shubham; Poolman, Bert
      Nature Communications (2019), 10 (1), 1-13CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      One of the grand challenges in chem. is the construction of functional out-of-equil. networks, which are typical of living cells. Building such a system from mol. components requires control over the formation and degrdn. of the interacting chems. and homeostasis of the internal phys.-chem. conditions. The provision and consumption of ATP lies at the heart of this challenge. Here we report the in vitro construction of a pathway in vesicles for sustained ATP prodn. that is maintained away from equil. by control of energy dissipation. We maintain a const. level of ATP with varying load on the system. The pathway enables us to control the transmembrane fluxes of osmolytes and to demonstrate basic physicochem. homeostasis. Our work demonstrates metabolic energy conservation and cell vol. regulatory mechanisms in a cell-like system at a level of complexity minimally needed for life.
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    11. 11
      Sikkema, H. R.; Gaastra, B. F.; Pols, T.; Poolman, B. Cell Fuelling and Metabolic Energy Conservation in Synthetic Cells. ChemBioChem. 2019, 20 (20), 2581– 2592,  DOI: 10.1002/cbic.201900398
      11
      Cell Fuelling and Metabolic Energy Conservation in Synthetic Cells
      Sikkema, Hendrik R.; Gaastra, Bauke F.; Pols, Tjeerd; Poolman, Bert
      ChemBioChem (2019), 20 (20), 2581-2592CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. We are aiming for a blue print for synthesizing (moderately complex) subcellular systems from mol. components and ultimately for constructing life. However, without comprehensive instructions and design principles, we rely on simple reaction routes to operate the essential functions of life. The first forms of synthetic life will not make every building block for polymers de novo according to complex pathways, rather they will be fed with amino acids, fatty acids and nucleotides. Controlled energy supply is crucial for any synthetic cell, no matter how complex. Herein, we describe the simplest pathways for the efficient generation of ATP and electrochem. ion gradients. We have estd. the demand for ATP by polymer synthesis and maintenance processes in small cell-like systems, and we describe circuits to control the need for ATP. We also present fluorescence-based sensors for pH, ionic strength, excluded vol., ATP/ADP, and viscosity, which allow the major physicochem. conditions inside cells to be monitored and tuned.
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    12. 12
      Bailoni, E.; Poolman, B. ATP Recycling Fuels Sustainable Glycerol 3 - Phosphate Formation in Synthetic Cells Fed by Dynamic Dialysis. ACS Synth. Biol. 2022, 11 (7), 2348– 2360,  DOI: 10.1021/acssynbio.2c00075
      12
      ATP Recycling Fuels Sustainable Glycerol 3-Phosphate Formation in Synthetic Cells Fed by Dynamic Dialysis
      Bailoni, Eleonora; Poolman, Bert
      ACS Synthetic Biology (2022), 11 (7), 2348-2360CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      The bottom-up construction of an autonomously growing, self-reproducing cell represents a great challenge for synthetic biol. Synthetic cellular systems are envisioned as out-of-equil. enzymic networks encompassed by a selectively open phospholipid bilayer allowing for protein-mediated communication; internal metabolite recycling is another key aspect of a sustainable metab. Importantly, gaining tight control over the external medium is essential to avoid thermodn. equil. due to nutrient depletion or waste buildup in a closed compartment (e.g., a test tube). Implementing a sustainable strategy for phospholipid biosynthesis is key to expanding the cellular boundaries. However, phospholipid biosynthesis is currently limited by substrate availability, e.g., of glycerol 3-phosphate, the essential core of phospholipid headgroups. Here, we reconstitute an enzymic network for sustainable glycerol 3-phosphate synthesis inside large unilamellar vesicles. We exploit the Escherichia coli glycerol kinase GlpK to synthesize glycerol 3-phosphate from externally supplied glycerol. We fuel phospholipid headgroup formation by sustainable L-arginine breakdown. In addn., we design and characterize a dynamic dialysis setup optimized for synthetic cells, which is used to control the external medium compn. and to achieve sustainable glycerol 3-phosphate synthesis.
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    13. 13
      Lee, K. Y.; Park, S. J.; Lee, K. A.; Kim, S. H.; Kim, H.; Meroz, Y.; Mahadevan, L.; Jung, K. H.; Ahn, T. K.; Parker, K. K.; Shin, K. Photosynthetic Artificial Organelles Sustain and Control ATP-Dependent Reactions in a Protocellular System. Nat. Biotechnol. 2018, 36 (6), 530– 535,  DOI: 10.1038/nbt.4140
      13
      Photosynthetic artificial organelles sustain and control ATP-dependent reactions in a protocellular system
      Lee, Keel Yong; Park, Sung-Jin; Lee, Keon Ah; Kim, Se-Hwan; Kim, Heeyeon; Meroz, Yasmine; Mahadevan, L.; Jung, Kwang-Hwan; Ahn, Tae Kyu; Parker, Kevin Kit; Shin, Kwanwoo
      Nature Biotechnology (2018), 36 (6), 530-535CODEN: NABIF9; ISSN:1087-0156. (Nature Research)
      Inside cells, complex metabolic reactions are distributed across the modular compartments of organelles. Reactions in organelles have been recapitulated in vitro by reconstituting functional protein machineries into membrane systems. However, maintaining and controlling these reactions is challenging. Here we designed, built, and tested a switchable, light-harvesting organelle that provides both a sustainable energy source and a means of directing intravesicular reactions. An ATP (ATP) synthase and two photoconverters (plant-derived photosystem II and bacteria-derived proteorhodopsin) enable ATP synthesis. Independent optical activation of the two photoconverters allows dynamic control of ATP synthesis: red light facilitates and green light impedes ATP synthesis. We encapsulated the photosynthetic organelles in a giant vesicle to form a protocellular system and demonstrated optical control of two ATP-dependent reactions, carbon fixation and actin polymn., with the latter altering outer vesicle morphol. Switchable photosynthetic organelles may enable the development of biomimetic vesicle systems with regulatory networks that exhibit homeostasis and complex cellular behaviors.
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    14. 14
      Partipilo, M.; Ewins, E. J.; Frallicciardi, J.; Robinson, T.; Poolman, B.; Slotboom, D. J. Minimal Pathway for the Regeneration of Redox Cofactors. JACS Au 2021, 1 (12), 2280– 2293,  DOI: 10.1021/jacsau.1c00406
      14
      Minimal Pathway for the Regeneration of Redox Cofactors
      Partipilo, Michele; Ewins, Eleanor J.; Frallicciardi, Jacopo; Robinson, Tom; Poolman, Bert; Slotboom, Dirk Jan
      JACS Au (2021), 1 (12), 2280-2293CODEN: JAAUCR; ISSN:2691-3704. (American Chemical Society)
      Effective metabolic pathways are essential for the construction of in vitro systems mimicking the biochem. complexity of living cells. Such pathways require the inclusion of a metabolic branch that ensures the availability of reducing equiv. Here, we built a minimal enzymic pathway confinable in the lumen of liposomes, in which the redox status of the nicotinamide cofactors NADH and NADPH is controlled by an externally provided formate. Formic acid permeates the membrane where a luminal formate dehydrogenase uses NAD+ to form NADH and carbon dioxide. Carbon dioxide diffuses out of the liposomes, leaving only the reducing equiv. in the lumen. A sol. transhydrogenase subsequently utilizes NADH for redn. of NADP+ thereby making NAD+ available again for the first reaction. The pathway is functional in liposomes ranging from a few hundred nanometers in diam. (large unilamellar vesicles) up to several tens of micrometers (giant unilamellar vesicles) and remains active over a period of 7 days. We demonstrate that the downstream biochem. process of redn. of glutathione disulfide can be driven by the transfer of reducing equiv. from formate via NAD(P)H, thereby providing a versatile set of electron donors for reductive metab.
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    15. 15
      Camps, M.; Herman, A.; Loh, E.; Loeb, L. A. Genetic Constraints on Protein Evolution. Crit. Rev. Biochem. Mol. Biol. 2007, 42 (5), 313– 326,  DOI: 10.1080/10409230701597642
      15
      Genetic constraints on protein evolution
      Camps, Manel; Herman, Asael; Loh, Ern; Loeb, Lawrence A.
      Critical Reviews in Biochemistry and Molecular Biology (2007), 42 (5), 313-326CODEN: CRBBEJ; ISSN:1040-9238. (Informa Healthcare)
      A review. Evolution requires the generation and optimization of new traits ("adaptation") and involves the selection of mutations that improve cellular function. These mutations are assumed to arise by selection of neutral mutations present at all times in the population. Here, the authors review recent evidence that indicates that deleterious mutations are more frequent in the population than previously recognized and that these mutations play a significant role in protein evolution through continuous pos. selection. Pos. selected mutations include adaptive mutations, i.e., mutations that directly affect enzymic function, and compensatory mutations, which suppress the pleiotropic effects of adaptive mutations. Compensatory mutations are by far the most frequent of the 2 and would allow potentially adaptive but deleterious mutations to persist long enough in the population to be pos. selected during episodes of adaptation. Compensatory mutations are, by definition, context-dependent and thus constrain the paths available for evolution. This provides a mechanistic basis for the examples of highly constrained evolutionary landscapes and parallel evolution reported in natural and exptl. populations. The present review article describes these recent advances in the field of protein evolution and discusses their implications for understanding the genetic basis of disease and for protein engineering in vitro.
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    16. 16
      Coombes, D.; Moir, J. W. B.; Poole, A. M.; Cooper, T. F.; Dobson, R. C. J. The Fitness Challenge of Studying Molecular Adaptation. Biochem. Soc. Trans. 2019, 47 (5), 1533– 1542,  DOI: 10.1042/BST20180626
      16
      The fitness challenge of studying molecular adaptation
      Coombes, David; Moir, James W. B.; Poole, Anthony M.; Cooper, Tim F.; Dobson, Renwick C. J.
      Biochemical Society Transactions (2019), 47 (5), 1533-1542CODEN: BCSTB5; ISSN:0300-5127. (Portland Press Ltd.)
      A review. Advances in bioinformatics and high-throughput genetic anal. increasingly allow us to predict the genetic basis of adaptive traits. These predictions can be tested and confirmed, but the mol.-level changes - i.e. the mol. adaptation - that link genetic differences to organism fitness remain generally unknown. In recent years, a series of studies have started to unpick the mechanisms of adaptation at the mol. level. In particular, this work has examd. how changes in protein function, activity, and regulation cause improved organismal fitness. Key to addressing mol. adaptations is identifying systems and designing expts. that integrate changes in the genome, protein chem. (mol. phenotype), and fitness. Knowledge of the mol. changes underpinning adaptations allow new insight into the constraints on, and repeatability of adaptations, and of the basis of non-additive interactions between adaptive mutations. Here we critically discuss a series of studies that examine the mol.-level adaptations that connect genetic changes and fitness.
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    17. 17
      Schmidt, S.; Sunyaev, S.; Bork, P.; Dandekar, T. Metabolites: A Helping Hand for Pathway Evolution?. Trends Biochem. Sci. 2003, 28 (6), 336– 341,  DOI: 10.1016/S0968-0004(03)00114-2
      17
      Metabolites: a helping hand for pathway evolution?
      Schmidt, Steffen; Sunyaev, Shamil; Bork, Peer; Dandekar, Thomas
      Trends in Biochemical Sciences (2003), 28 (6), 336-341CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Science Ltd.)
      A review. The evolution of enzymes and pathways is under debate. Recent studies show that recruitment of single enzymes from different pathways could be the driving force for pathway evolution. Other mechanisms of evolution, such as pathway duplication, enzyme specialization, de novo invention of pathways or retro-evolution of pathways, appear to be less abundant. Twenty percent of enzyme superfamilies are quite variable, not only in changing reaction chem. or metabolite type but in changing both at the same time. These variable superfamilies account for nearly half of all known reactions. The most frequently occurring metabolites provide a helping hand for such changes because they can be accommodated by many enzyme superfamilies. Thus, a picture is emerging in which new pathways are evolving from central metabolites by preference, thereby keeping the overall topol. of the metabolic network.
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    18. 18
      Pfeiffer, T.; Soyer, O. S.; Bonhoeffer, S. The Evolution of Connectivity in Metabolic Networks. PLoS Biol. 2005, 3 (7), e228,  DOI: 10.1371/journal.pbio.0030228
      There is no corresponding record for this reference.
    19. 19
      Sajed, T.; Marcu, A.; Ramirez, M.; Pon, A.; Guo, A. C.; Knox, C.; Wilson, M.; Grant, J. R.; Djoumbou, Y.; Wishart, D. S. ECMDB 2.0: A Richer Resource for Understanding the Biochemistry of E. Coli. Nucleic Acids Res. 2016, 44 (D1), D495– D501,  DOI: 10.1093/nar/gkv1060
      19
      ECMDB 2.0: a richer resource for understanding the biochemistry of E. coli
      Sajed, Tanvir; Marcu, Ana; Ramirez, Miguel; Pon, Allison; Guo, An Chi; Knox, Craig; Wilson, Michael; Grant, Jason R.; Djoumbou, Yannick; Wishart, David S.
      Nucleic Acids Research (2016), 44 (D1), D495-D501CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      ECMDB or the Escherichia coli metabolome database is a comprehensive database contg. detailed information about the genome and metabolome of E. coli (K-12). First released in 2012, the ECMDB has undergone substantial expansion and many modifications over the past 4 years. This manuscript describes the most recent version of ECMDB (ECMDB 2.0). In particular, it provides a comprehensive update of the database that was previously described in the 2013 NAR Database Issue and details many of the addns. and improvements made to the ECMDB over that time. Some of the most important or significant enhancements include a 13-fold increase in the no. of metabolic pathway diagrams (from 125 to 1650), a 3-fold increase in the no. of compds. linked to pathways (from 1058 to 3280), the addn. of dozens of operon/metabolite signaling pathways, a 44% increase in the no. of compds. in the database (from 2610 to 3760), a 7-fold increase in the no. of compds. with NMR or MS spectra (from 412 to 3261) and a massive increase in the no. of external links to other E. coli or chem. resources. These addns., along with many other enhancements aimed at improving the ease or speed of querying, searching and viewing the data within ECMDB should greatly facilitate the understanding of not only the metab. of E. coli, but also allow the in-depth exploration of its extensive metabolic networks, its many signaling pathways and its essential biochem.
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    20. 20
      Blank, L. M.; Ebert, B. E.; Buehler, K.; Bühler, B. Redox Biocatalysis and Metabolism: Molecular Mechanisms and Metabolic Network Analysis. Antioxidants Redox Signal. 2010, 13 (3), 349– 394,  DOI: 10.1089/ars.2009.2931
      20
      Redox biocatalysis and metabolism: molecular mechanisms and metabolic network analysis
      Blank Lars M; Ebert Birgitta E; Buehler Katja; Buhler Bruno
      Antioxidants & redox signaling (2010), 13 (3), 349-94 ISSN:.
      Whole-cell biocatalysis utilizes native or recombinant enzymes produced by cellular metabolism to perform synthetically interesting reactions. Besides hydrolases, oxidoreductases represent the most applied enzyme class in industry. Oxidoreductases are attributed a high future potential, especially for applications in the chemical and pharmaceutical industries, as they enable highly interesting chemistry (e.g., the selective oxyfunctionalization of unactivated C-H bonds). Redox reactions are characterized by electron transfer steps that often depend on redox cofactors as additional substrates. Their regeneration typically is accomplished via the metabolism of whole-cell catalysts. Traditionally, studies towards productive redox biocatalysis focused on the biocatalytic enzyme, its activity, selectivity, and specificity, and several successful examples of such processes are running commercially. However, redox cofactor regeneration by host metabolism was hardly considered for the optimization of biocatalytic rate, yield, and/or titer. This article reviews molecular mechanisms of oxidoreductases with synthetic potential and the host redox metabolism that fuels biocatalytic reactions with redox equivalents. The tools discussed in this review for investigating redox metabolism provide the basis for studies aiming at a deeper understanding of the interplay between synthetically active enzymes and metabolic networks. The ultimate goal of rational whole-cell biocatalyst engineering and use for fine chemical production is discussed.
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    21. 21
      Agledal, L.; Niere, M.; Ziegler, M. The Phosphate Makes a Difference: Cellular Functions of NADP. Redox Rep. 2010, 15 (1), 2– 10,  DOI: 10.1179/174329210X12650506623122
      21
      The phosphate makes a difference: cellular functions of NADP
      Agledal, Line; Niere, Marc; Ziegler, Mathias
      Redox Report (2010), 15 (1), 2-10CODEN: RDRPE4; ISSN:1351-0002. (Maney Publishing)
      A review. Recent research has unraveled a no. of unexpected functions of the pyridine nucleotides. In this review, we will highlight the variety of known physiol. roles of NADP. In its reduced form (NADPH), this mol. represents a universal electron donor, not only to drive biosynthetic pathways. Perhaps even more importantly, NADPH is the unique provider of reducing equiv. to maintain or regenerate the cellular detoxifying and antioxidative defense systems. The roles of NADPH in redox sensing and as substrate for NADPH oxidases to generate reactive oxygen species further extend its scope of functions. NADP+, on the other hand, has acquired signaling functions. Its conversion to second messengers in calcium signaling may have crit. impact on important cellular processes. The generation of NADP by NAD kinases is a key determinant of the cellular NADP concn. The regulation of these enzymes may, therefore, be crit. to feed the diversity of NADP-dependent processes adequately. The increasing recognition of the multiple roles of NADP has thus led to exciting new insights in this expanding field.
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    22. 22
      Spaans, S. K.; Weusthuis, R. A.; van der Oost, J.; Kengen, S. W. M. NADPH-Generating Systems in Bacteria and Archaea. Front. Microbiol. 2015, 6, 742,  DOI: 10.3389/fmicb.2015.00742
      22
      NADPH-generating systems in bacteria and archaea
      Spaans Sebastiaan K; van der Oost John; Kengen Serve W M; Weusthuis Ruud A
      Frontiers in microbiology (2015), 6 (), 742 ISSN:1664-302X.
      Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components and many products in biotechnology. The efficient synthesis of many of these products, however, is limited by the rate of NADPH regeneration. Hence, a thorough understanding of the reactions involved in the generation of NADPH is required to increase its turnover through rational strain improvement. Traditionally, the main engineering targets for increasing NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA) cycle. However, the importance of alternative NADPH-generating reactions has recently become evident. In the current review, the major canonical and non-canonical reactions involved in the production and regeneration of NADPH in prokaryotes are described, and their key enzymes are discussed. In addition, an overview of how different enzymes have been applied to increase NADPH availability and thereby enhance productivity is provided.
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    23. 23
      Fuhrer, T.; Sauer, U. Different Biochemical Mechanisms Ensure Network-Wide Balancing of Reducing Equivalents in Microbial Metabolism. J. Bacteriol. 2009, 191 (7), 2112– 2121,  DOI: 10.1128/JB.01523-08
      23
      Different biochemical mechanisms ensure network-wide balancing of reducing equivalents in microbial metabolism
      Fuhrer, Tobias; Sauer, Uwe
      Journal of Bacteriology (2009), 191 (7), 2112-2121CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      To sustain growth, the catabolic formation of the redox equiv. NADPH must be balanced with the anabolic demand. The mechanisms that ensure such network-wide balancing, however, are presently not understood. Based on 13C-detected intracellular fluxes, metabolite concns., and cofactor specificities for all relevant central metabolic enzymes, we have quantified catabolic NADPH prodn. in Agrobacterium tumefaciens, Bacillus subtilis, Escherichia coli, Paracoccus versutus, Pseudomonas fluorescens, Rhodobacter sphaeroides, Sinorhizobium meliloti, and Zymomonas mobilis. For six species, the estd. NADPH prodn. from glucose catabolism exceeded the requirements for biomass synthesis. Exceptions were P. fluorescens, with balanced rates, and E. coli, with insufficient catabolic prodn., in which about one-third of the NADPH is supplied via the membrane-bound transhydrogenase PntAB. P. versutus and B. subtilis were the only species that appear to rely on transhydrogenases for balancing NADPH overprodn. during growth on glucose. In the other four species, the main but not exclusive redox-balancing mechanism appears to be the dual cofactor specificities of several catabolic enzymes and/or the existence of isoenzymes with distinct cofactor specificities, in particular glucose 6-phosphate dehydrogenase. An unexpected key finding for all species, except E. coli and B. subtilis, was the lack of cofactor specificity in the oxidative pentose phosphate pathway, which contrasts with the textbook view of the pentose phosphate pathway dehydrogenases as being NADP+ dependent.
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    24. 24
      Goldford, J. E.; George, A. B.; Flamholz, A. I.; Segre, D. Protein Cost Minimization Promotes the Emergence of Coenzyme Redundancy. Proc. Natl. Acad. Sci. U. S. A. 2022, 119 (14), e2110787119,  DOI: 10.1073/pnas.2110787119
      24
      Protein cost minimization promotes the emergence of coenzyme redundancy
      Goldford, Joshua E.; George, Ashish B.; Flamholz, Avi I.; Segre, Daniel
      Proceedings of the National Academy of Sciences of the United States of America (2022), 119 (14), e2110787119CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Coenzymes distribute a variety of chem. moieties throughout cellular metab., participating in group (e.g., phosphate and acyl) and electron transfer. For a variety of reactions requiring acceptors or donors of specific resources, there often exist degenerate sets of mols. [e.g., NAD(H) and NADP(H)] that carry out similar functions. Although the physiol. roles of various coenzyme systems are well established, it is unclear what selective pressures may have driven the emergence of coenzyme redundancy. Here, we use genome-wide metabolic modeling approaches to decomp. the selective pressures driving enzymic specificity for either NAD(H) or NADP(H) in the metabolic network of Escherichia coli. We found that few enzymes are thermodynamically constrained to using a single coenzyme, and in principle a metabolic network relying on only NAD(H) is feasible. However, structural and sequence analyses revealed widespread conservation of residues that retain selectivity for either NAD(H) or NADP(H), suggesting that addnl. forces may shape specificity. Using a model accounting for the cost of oxidoreductase enzyme expression, we found that coenzyme redundancy universally reduces the minimal amt. of protein required to catalyze coenzyme-coupled reactions, inducing individual reactions to strongly prefer one coenzyme over another when reactions are near thermodn. equil. We propose that protein minimization generically promotes coenzyme redundancy and that coenzymes typically thought to exist in a single pool (e.g., CoA [CoA]) may exist in more than one form (e.g., dephospho-CoA).
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    25. 25
      Xiao, W.; Wang, R. S.; Handy, D. E.; Loscalzo, J. NAD(H) and NADP(H) Redox Couples and Cellular Energy Metabolism. Antioxidants Redox Signal. 2018, 28 (3), 251– 272,  DOI: 10.1089/ars.2017.7216
      25
      NAD(H) and NADP(H) Redox Couples and Cellular Energy Metabolism
      Xiao Wusheng; Wang Rui-Sheng; Handy Diane E; Loscalzo Joseph
      Antioxidants & redox signaling (2018), 28 (3), 251-272 ISSN:.
      SIGNIFICANCE: The nicotinamide adenine dinucleotide (NAD(+))/reduced NAD(+) (NADH) and NADP(+)/reduced NADP(+) (NADPH) redox couples are essential for maintaining cellular redox homeostasis and for modulating numerous biological events, including cellular metabolism. Deficiency or imbalance of these two redox couples has been associated with many pathological disorders. Recent Advances: Newly identified biosynthetic enzymes and newly developed genetically encoded biosensors enable us to understand better how cells maintain compartmentalized NAD(H) and NADP(H) pools. The concept of redox stress (oxidative and reductive stress) reflected by changes in NAD(H)/NADP(H) has increasingly gained attention. The emerging roles of NAD(+)-consuming proteins in regulating cellular redox and metabolic homeostasis are active research topics. CRITICAL ISSUES: The biosynthesis and distribution of cellular NAD(H) and NADP(H) are highly compartmentalized. It is critical to understand how cells maintain the steady levels of these redox couple pools to ensure their normal functions and simultaneously avoid inducing redox stress. In addition, it is essential to understand how NAD(H)- and NADP(H)-utilizing enzymes interact with other signaling pathways, such as those regulated by hypoxia-inducible factor, to maintain cellular redox homeostasis and energy metabolism. FUTURE DIRECTIONS: Additional studies are needed to investigate the inter-relationships among compartmentalized NAD(H)/NADP(H) pools and how these two dinucleotide redox couples collaboratively regulate cellular redox states and cellular metabolism under normal and pathological conditions. Furthermore, recent studies suggest the utility of using pharmacological interventions or nutrient-based bioactive NAD(+) precursors as therapeutic interventions for metabolic diseases. Thus, a better understanding of the cellular functions of NAD(H) and NADP(H) may facilitate efforts to address a host of pathological disorders effectively. Antioxid. Redox Signal. 28, 251-272.
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    26. 26
      Breuer, M.; Earnest, T. M.; Merryman, C.; Wise, K. S.; Sun, L.; Lynott, M. R.; Hutchison, C. A.; Smith, H. O.; Lapek, J. D.; Gonzalez, D. J.; Crécy-Lagard, V. de; Haas, D.; Hanson, A. D.; Labhsetwar, P.; Glass, J. I.; Luthey-Schulten, Z. Essential Metabolism for a Minimal Cell. eLife 2019, 8, e36842,  DOI: 10.7554/eLife.36842
      There is no corresponding record for this reference.
    27. 27
      Wichmann, R.; Vasic-Racki, D. Cofactor Regeneration at the Lab Scale. Adv. Biochem. Eng. Biotechnol. 2005, 92, 225– 260,  DOI: 10.1007/b98911
      27
      Cofactor regeneration at the lab scale
      Wichmann, R.; Vasic-Racki, D.
      Advances in Biochemical Engineering/Biotechnology (2005), 92 (Technology Transfer in Biotechnology), 225-260CODEN: ABEBDZ; ISSN:0724-6145. (Springer GmbH)
      A review. Progress made in lab.-scale applications of various coenzyme regeneration systems over the last two decades has mainly focused on the applications of NAD+/NADH- and NADP+/NADPH-dependent oxidoreductase reactions. In situ regeneration systems for these reactions, as well as whole cell, enzymic, electro-enzymic, chem., and photochem. reactions are presented, including details about their efficiency and novelty. The progress of enzyme reaction engineering is also reported.
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    28. 28
      Weckbecker, A.; Gröger, H.; Hummel, W. Regeneration of Nicotinamide Coenzymes: Principles and Applications for the Synthesis of Chiral Compounds. Biosyst. Eng. I 2010, 195– 242,  DOI: 10.1007/10_2009_55
      There is no corresponding record for this reference.
    29. 29
      Shi, T.; Han, P.; You, C.; Zhang, Y. H. P. J. An in Vitro Synthetic Biology Platform for Emerging Industrial Biomanufacturing: Bottom-up Pathway Design. Synth. Syst. Biotechnol. 2018, 3 (3), 186– 195,  DOI: 10.1016/j.synbio.2018.05.002
      29
      An in vitro synthetic biology platform for emerging industrial biomanufacturing: Bottom-up pathway design
      Shi Ting; Han Pingping; You Chun; Zhang Yi-Heng P Job
      Synthetic and systems biotechnology (2018), 3 (3), 186-195 ISSN:.
      Although most in vitro (cell-free) synthetic biology projects are usually used for the purposes of fundamental research or the formation of high-value products, in vitro synthetic biology platform, which can implement complicated biochemical reactions by the in vitro assembly of numerous enzymes and coenzymes, has been proposed for low-cost biomanufacturing of bioenergy, food, biochemicals, and nutraceuticals. In addition to the most important advantage-high product yield, in vitro synthetic biology platform features several other biomanufacturing advantages, such as fast reaction rate, easy product separation, open process control, broad reaction condition, tolerance to toxic substrates or products, and so on. In this article, we present the basic bottom-up design principles of in vitro synthetic pathway from basic building blocks-BioBricks (thermoenzymes and/or immobilized enzymes) to building modules (e.g., enzyme complexes or multiple enzymes as a module) with specific functions. With development in thermostable building blocks-BioBricks and modules, the in vitro synthetic biology platform would open a new biomanufacturing age for the cost-competitive production of biocommodities.
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    30. 30
      Tassano, E.; Hall, M. Enzymatic Self-Sufficient Hydride Transfer Processes. Chem. Soc. Rev. 2019, 48 (23), 5596– 5615,  DOI: 10.1039/C8CS00903A
      30
      Enzymatic self-sufficient hydride transfer processes
      Tassano, Erika; Hall, Melanie
      Chemical Society Reviews (2019), 48 (23), 5596-5615CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. A no. of self-sufficient hydride transfer processes have been reported in biocatalysis, with a common feature being the dependence on nicotinamide as a cofactor. This cofactor is provided in catalytic amts. and serves as a hydride shuttle to connect two or more enzymic redox events, usually ensuring overall redox neutrality. Creative systems were designed to produce synthetic sequences characterized by high hydride economy, typically going in hand with excellent atom economy. Several redox enzymes have been successfully combined in one-pot one-step to allow functionalization of a large variety of mols. while preventing byproduct formation. This review analyzes and classifies the various strategies, with a strong focus on efficiency, which is evaluated here in terms of the hydride economy and measured by the turnover no. of the nicotinamide cofactor(s). The review ends with a crit. evaluation of the reported systems and highlights areas where further improvements might be desirable.
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    31. 31
      Dekker, J. P.; Boekema, E. J. Supramolecular Organization of Thylakoid Membrane Proteins in Green Plants. Biochim. Biophys. Acta - Bioenerg. 2005, 1706 (1–2), 12– 39,  DOI: 10.1016/j.bbabio.2004.09.009
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      Supramolecular organization of thylakoid membrane proteins in green plants
      Dekker, Jan P.; Boekema, Egbert J.
      Biochimica et Biophysica Acta, Bioenergetics (2005), 1706 (1-2), 12-39CODEN: BBBEB4; ISSN:0005-2728. (Elsevier B.V.)
      A review. The light reactions of photosynthesis in green plants are mediated by four large protein complexes, embedded in the thylakoid membrane of the chloroplast. Photosystem I (PSI) and Photosystem II (PSII) are both organized into large supercomplexes with variable amts. of membrane-bound peripheral antenna complexes. PSI consists of a monomeric core complex with single copies of four different LHCI proteins and has binding sites for addnl. LHCI and/or LHCII complexes. PSII supercomplexes are dimeric and contain usually two to four copies of trimeric LHCII complexes. These supercomplexes have a further tendency to assoc. into megacomplexes or into cryst. domains, of which several types have been characterized. Together with the specific lipid compn., the structural features of the main protein complexes of the thylakoid membranes form the main trigger for the segregation of PSII and LHCII from PSI and ATPase into stacked grana membranes. We suggest that the margins, the strongly folded regions of the membranes that connect the grana, are essentially protein-free, and that protein-protein interactions in the lumen also det. the shape of the grana. We also discuss which mechanisms det. the stacking of the thylakoid membranes and how the supramol. organization of the pigment-protein complexes in the thylakoid membrane and their flexibility may play roles in various regulatory mechanisms of green plant photosynthesis.
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    32. 32
      Thauer, R. K.; Jungermann, K.; Decker, K. Energy Conservation in Chemotrophic Anaerobic Bacteria. Bacteriol. Rev. 1977, 41 (1), 100– 180,  DOI: 10.1128/br.41.1.100-180.1977
      32
      Energy conservation in chemotrophic anaerobic bacteria
      Thauer, Rudolf K.; Jungermann, Kurt; Decker, Karl
      Bacteriological Reviews (1977), 41 (1), 100-80CODEN: BAREA8; ISSN:0005-3678.
      A review with 743 refs.
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    33. 33
      Lehninger, A. L.; Lehninger Principles of Biochemistry; Macmillan, 2005.
      There is no corresponding record for this reference.
    34. 34
      Relyea, H. A.; Van Der Donk, W. A. Mechanism and Applications of Phosphite Dehydrogenase. Bioorg. Chem. 2005, 33 (3), 171– 189,  DOI: 10.1016/j.bioorg.2005.01.003
      34
      Mechanism and applications of phosphite dehydrogenase
      Relyea, Heather A.; van der Donk, Wilfred A.
      Bioorganic Chemistry (2005), 33 (3), 171-189CODEN: BOCMBM; ISSN:0045-2068. (Elsevier)
      A review. Phosphite dehydrogenase catalyzes the NAD+-dependent oxidn. of hydrogen phosphonate (common name phosphite) to phosphate in what amts. to a formal phosphoryl transfer reaction from hydride to hydroxide. This review places the enzyme in the context of phosphorus redox metab. in nature and discusses the results of mechanistic investigations into its reaction mechanism. The potential of the enzyme as a NAD(P)H cofactor regeneration system is discussed as well as efforts to engineer the cofactor specificity of the protein.
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    35. 35
      Wu, J.; Huang, Y.; Ye, W.; Li, Y. CO2 Reduction: From the Electrochemical to Photochemical Approach. Adv. Sci. 2017, 4 (11), 1700194,  DOI: 10.1002/advs.201700194
      35
      CO2 Reduction: From the Electrochemical to Photochemical Approach
      Wu Jinghua; Huang Yang; Ye Wen; Li Yanguang
      Advanced science (Weinheim, Baden-Wurttemberg, Germany) (2017), 4 (11), 1700194 ISSN:2198-3844.
      Increasing CO2 concentration in the atmosphere is believed to have a profound impact on the global climate. To reverse the impact would necessitate not only curbing the reliance on fossil fuels but also developing effective strategies capture and utilize CO2 from the atmosphere. Among several available strategies, CO2 reduction via the electrochemical or photochemical approach is particularly attractive since the required energy input can be potentially supplied from renewable sources such as solar energy. In this Review, an overview on these two different but inherently connected approaches is provided and recent progress on the development, engineering, and understanding of CO2 reduction electrocatalysts and photocatalysts is summarized. First, the basic principles that govern electrocatalytic or photocatalytic CO2 reduction and their important performance metrics are discussed. Then, a detailed discussion on different CO2 reduction electrocatalysts and photocatalysts as well as their generally designing strategies is provided. At the end of this Review, perspectives on the opportunities and possible directions for future development of this field are presented.
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    36. 36
      Beber, M. E.; Gollub, M. G.; Mozaffari, D.; Shebek, K. M.; Flamholz, A. I.; Milo, R.; Noor, E. EQuilibrator 3.0: A Database Solution for Thermodynamic Constant Estimation. Nucleic Acids Res. 2022, 50 (D1), D603– D609,  DOI: 10.1093/nar/gkab1106
      36
      The eQuilibrator 3.0: a database solution for thermodynamic constant estimation
      Beber, Moritz E.; Gollub, Mattia G.; Mozaffari, Dana; Shebek, Kevin M.; Flamholz, Avi I.; Milo, Ron; Noor, Elad
      Nucleic Acids Research (2022), 50 (D1), D603-D609CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      EQuilibrator (equilibrator.weizmann.ac.il) is a database of biochem. equil. consts. and Gibbs free energies, originally designed as a web-based interface. While the website now counts around 1,000 distinct monthly users, its design could not accommodate larger compd. databases and it lacked a scalable Application Programming Interface (API) for integration into other tools developed by the systems biol. community. Here, we report on the recent updates to the database as well as the addn. of a new Python-based interface to eQuilibrator that adds many new features such as a 100-fold larger compd. database, the ability to add novel compds., improvements in speed and memory use, and correction for Mg2+ ion concns. Moreover, the new interface can compute the covariance matrix of the uncertainty between ests., for which we show the advantages and describe the application in metabolic modeling. We foresee that these improvements will make thermodn. modeling more accessible and facilitate the integration of eQuilibrator into other software platforms.
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    37. 37
      Cho, A.; Yun, H.; Park, J. H.; Lee, S. Y.; Park, S. Prediction of Novel Synthetic Pathways for the Production of Desired Chemicals. BMC Syst. Biol. 2010, 4 (1), 1– 16,  DOI: 10.1186/1752-0509-4-35
      There is no corresponding record for this reference.
    38. 38
      Morgado, G.; Gerngross, D.; Roberts, T. M.; Panke, S. Synthetic Biology for Cell-Free Biosynthesis: Fundamentals of Designing Novel in Vitro Multi-Enzyme Reaction Networks. Synth. Biol. Eng. 2016, 162, 117– 146,  DOI: 10.1007/10_2016_13
      There is no corresponding record for this reference.
    39. 39
      Noor, E.; Bar-Even, A.; Flamholz, A.; Reznik, E.; Liebermeister, W.; Milo, R. Pathway Thermodynamics Highlights Kinetic Obstacles in Central Metabolism. PLoS Comput. Biol. 2014, 10 (2), e1003483,  DOI: 10.1371/journal.pcbi.1003483
      39
      Pathway thermodynamics highlights kinetic obstacles in central metabolism
      Noor, Elad; Bar-Even, Arren; Flamholz, Avi; Reznik, Ed; Liebermeister, Wolfram; Milo, Ron
      PLoS Computational Biology (2014), 10 (2), e1003483/1-e1003483/12, 12 pp.CODEN: PCBLBG; ISSN:1553-7358. (Public Library of Science)
      In metab. research, thermodn. is usually used to det. the directionality of a reaction or the feasibility of a pathway. However, the relationship between thermodn. potentials and fluxes is not limited to questions of directionality: thermodn. also affects the kinetics of reactions through the flux-force relationship, which states that the logarithm of the ratio between the forward and reverse fluxes is directly proportional to the change in Gibbs energy due to a reaction (ΔrG'). Accordingly, if an enzyme catalyzes a reaction with a ΔrG' of -5.7 kJ/mol then the forward flux will be roughly ten times the reverse flux. As ΔrG' approaches equil. (ΔrG' = 0 kJ/mol), exponentially more enzyme counterproductively catalyzes the reverse reaction, reducing the net rate at which the reaction proceeds. Thus, the enzyme level required to achieve a given flux increases dramatically near equil. Here, we develop a framework for quantifying the degree to which pathways suffer these thermodn. limitations on flux. For each pathway, we calc. a single thermodynamically-derived metric (the Max-min Driving Force, MDF), which enables objective ranking of pathways by the degree to which their flux is constrained by low thermodn. driving force. Our framework accounts for the effect of pH, ionic strength and metabolite concn. ranges and allows us to quantify how alterations to the pathway structure affect the pathway's thermodn. Applying this methodol. to pathways of central metab. sheds light on some of their features, including metabolic bypasses (e.g., fermn. pathways bypassing substrate-level phosphorylation), substrate channeling (e.g., of oxaloacetate from malate dehydrogenase to citrate synthase), and use of alternative cofactors (e.g., quinone as an electron acceptor instead of NAD). The methods presented here place another arrow in metabolic engineers' quiver, providing a simple means of evaluating the thermodn. and kinetic quality of different pathway chemistries that produce the same mols.
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    40. 40
      Guynn, R. W.; Gelberg, H. J.; Veech, R. L. Equilibrium Constants of the Malate Dehydrogenase, Citrate Synthase, Citrate Lyase, and Acetyl Coenzyme A Hydrolysis Reactions under Physiological Conditions. J. Biol. Chem. 1973, 248 (20), 6957– 6965,  DOI: 10.1016/S0021-9258(19)43346-2
      40
      Equilibrium constants of the malate dehydrogenase, citrate synthase, citrate lyase, and acetyl coenzyme A hydrolysis reactions under physiological conditions
      Guynn, Robert W.; Gelberg, Harris J.; Veech, Richard L.
      Journal of Biological Chemistry (1973), 248 (20), 6957-65CODEN: JBCHA3; ISSN:0021-9258.
      The obsd. equil. consts. (Kobs) of the malate dehydrogenase (EC 1.1.1.37), citrate synthase (EC 4.1.3.7), and citrate lyase (EC 4.1.3.6) reactions were detd. under near physiol. conditions (38°, pH 7.0, ionic strength 0.25, free [Mg2+] = 10-3M). From these values, the observed std. free energy change (ΔG0obs) for the hydrolysis of acetyl-CoA was detd. Under the above conditions, and taking the std. state of liq. water to have activity = unity, the equil. consts. of the 3 reactions at pH 7.0 were 2.86 × 10-5 for malate dehydrogenase, 2.24 × 106 for citrate synthase, and 2.22 M-1 for citrate lyase. The values obtained for Kobs of the citrate synthase and citrate lyase reactions vary to the same extent with the changes in Mg2+ concn. At free [Mg2+] = O, Kobs for the citrate synthase reaction is 1.01 × 106 and the Kobs for the citrate lyase reaction is 1.00M-1. In contrast, malate dehydrogenase is unaffected by the concn. of free Mg2+ up to 4 mM. From the consts. of the citrate synthase and citrate lyase reactions, the Kobs for the hydrolysis of acetyl-CoA under near physiol. conditions is calcd. to be 1.01 × 106M, corresponding to a free energy change of -8.54 kcal/mole (-35.75 kJ/mole) independent of the free Mg2+ concn.
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    41. 41
      Williamson, D. H.; Lund, P.; K, H. A. The Redox State of Nicotinamide Adenine Dinucleotide in the Cytoplasm and Mitochondria of Rat Liver. Biochem. J. 1967, 103 (2), 514,  DOI: 10.1042/bj1030514
      41
      Redox state of free nicotinamide adenine dinucleotide in the cytoplasm and mitochondria of rat liver
      Williamson, Dermot Hedley; Lund, Patricia; Krebs, Hans A.
      Biochemical Journal (1967), 103 (), 514-27CODEN: BIJOAK; ISSN:0264-6021.
      The concns. of the oxidized and reduced substrates of the lactate, β-hydroxybutyrate, and glutamate dehydrogenase systems were measured in rat livers freeze-clamped as soon as possible after death. The substrates of these dehydrogenases are likely to be in equil. with free NAD and NADH, and the ratio of the free dinucleotides can be calcd. from the measured concns. of the substrates and the equil. consts. (Holzer, et al., CA 51, 6776h; Bucher and Klingenberg, CA 53, 22147a). The lactate dehydrogenase system reflects the NAD-to-NADH ratio in the cytoplasm, the β-hydroxybutyrate dehydrogenase that in the mitochondrial cristae, and the glutamate dehydrogenase that in the mitochondrial matrix. The equil. consts. of lactate dehydrogenase (EC 1.1.1.27), β-hydroxybutyrate dehydrogenase (EC 1.1.1.30), and malate dehydrogenase (EC 1.1.1.37) were redetd. for near-physiol. conditions (38°; ionic strength 0.25). The mean NAD-to-NADH ratio of rat liver cytoplasm was calcd. as 725 (pH 7.0) in well fed rats, 528 in starved rats, and 208 in alloxan-diabetic rats. The NAD to-NADH ratio for the mitochondrial matrix and cristae gave virtually identical values in the same metabolic state. This indicates that β-hydroxybutyrate dehydrogenase and glutamate dehydrogenase share a common pool of dinucleotide. The mean NAD-to-NADH ratio within the liver mitochondria of well fed rats was ∼8. It fell to ∼5 in starvation and rose to ∼10 in alloxan-diabetes. The NAD-to-NADH ratios of cytoplasm and mitochondria are thus greatly different and do not necessarily move in parallel when the metabolic state of the liver changes. The ratios found for the free dinucleotides differ greatly from those recorded for the total dinucleotides because much more NADH than NAD is protein bound. The bearing of these findings on various problems, including the following, is discussed: the no. of NAD-NADH pools in liver cells; the applicability of the method to tissues other than liver; the transhydrogenase activity of glutamate dehydrogenase; the physiol. significance of the difference of the redox states of mitochondria and cytoplasm; aspects of the regulation of the redox state of cell compartments; the steady-state concn. of mitochondrial oxalacetate; the relations between the redox state of cell compartments and ketosis.
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    42. 42
      Bailoni, E.; Partipilo, M.; Coenradij, J.; Grundel, D. A. J.; Slotboom, D. J.; Poolman, B. Minimal Out-of-Equilibrium Metabolism for Synthetic Cells: A Membrane Perspective. ACS Synth. Biol. 2023,  DOI: 10.1021/acssynbio.3c00062
      There is no corresponding record for this reference.
    43. 43
      Crnković, A.; Srnko, M.; Anderluh, G. Biological Nanopores: Engineering on Demand. Life 2021, 11 (1), 27,  DOI: 10.3390/life11010027
      43
      Biological nanopores: engineering on demand
      Crnkovic, Ana; Srnko, Marija; Anderluh, Gregor
      Life (Basel, Switzerland) (2021), 11 (1), 27CODEN: LBSIB7; ISSN:2075-1729. (MDPI AG)
      Nanopore-based sensing is a powerful technique for the detection of diverse org. and inorg. mols., long-read sequencing of nucleic acids, and single-mol. analyses of enzymic reactions. Selected from natural sources, protein-based nanopores enable rapid, label-free detection of analytes. Furthermore, these proteins are easy to produce, form pores with defined sizes, and can be easily manipulated with std. mol. biol. techniques. The range of possible analytes can be extended by using externally added adapter mols. Here, we provide an overview of current nanopore applications with a focus on engineering strategies and solns.
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    44. 44
      Diallinas, G. Understanding Transporter Specificity and the Discrete Appearance of Channel-like Gating Domains in Transporters. Front. Pharmacol. 2014,  DOI: 10.3389/fphar.2014.00207
      There is no corresponding record for this reference.
    45. 45
      Van De Cauter, L.; Fanalista, F.; Van Buren, L.; De Franceschi, N.; Godino, E.; Bouw, S.; Danelon, C.; Dekker, C.; Koenderink, G. H.; Ganzinger, K. A. Optimized CDICE for Efficient Reconstitution of Biological Systems in Giant Unilamellar Vesicles. ACS Synth. Biol. 2021, 10 (7), 1690– 1702,  DOI: 10.1021/acssynbio.1c00068
      45
      Optimized cDICE for Efficient Reconstitution of Biological Systems in Giant Unilamellar Vesicles
      Van de Cauter, Lori; Fanalista, Federico; van Buren, Lennard; De Franceschi, Nicola; Godino, Elisa; Bouw, Sharon; Danelon, Christophe; Dekker, Cees; Koenderink, Gijsje H.; Ganzinger, Kristina A.
      ACS Synthetic Biology (2021), 10 (7), 1690-1702CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Giant unilamellar vesicles (GUVs) are often used to mimic biol. membranes in reconstitution expts. They are also widely used in research on synthetic cells, as they provide a mech. responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomols. in GUVs, there is no one-size-fits-all soln. and reliable GUV fabrication still remains a major exptl. hurdle in the field. Here, we show that defect-free GUVs contg. complex biochem. systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of biol. systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (protein synthesis using recombinant elements) system. Our optimized cDICE method displays promising potential to become a std. method in biophysics and bottom-up synthetic biol.
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    46. 46
      Adamala, K. P.; Martin-Alarcon, D. A.; Guthrie-Honea, K. R.; Boyden, E. S. Engineering Genetic Circuit Interactions within and between Synthetic Minimal Cells. Nat. Chem. 2017, 9 (5), 431– 439,  DOI: 10.1038/nchem.2644
      46
      Engineering genetic circuit interactions within and between synthetic minimal cells
      Adamala, Katarzyna P.; Martin-Alarcon, Daniel A.; Guthrie-Honea, Katriona R.; Boyden, Edward S.
      Nature Chemistry (2017), 9 (5), 431-439CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
      Genetic circuits and reaction cascades are of great importance for synthetic biol., biochem. and bioengineering. An open question is how to maximize the modularity of their design to enable the integration of different reaction networks and to optimize their scalability and flexibility. One option is encapsulation within liposomes, which enables chem. reactions to proceed in well-isolated environments. Here we adapt liposome encapsulation to enable the modular, controlled compartmentalization of genetic circuits and cascades. We demonstrate that it is possible to engineer genetic circuit-contg. synthetic minimal cells (synells) to contain multiple-part genetic cascades, and that these cascades can be controlled by external signals as well as inter-liposomal communication without crosstalk. We also show that liposomes that contain different cascades can be fused in a controlled way so that the products of incompatible reactions can be brought together. Synells thus enable a more modular creation of synthetic biol. cascades, an essential step towards their ultimate programmability.
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    47. 47
      Rampioni, G.; D’Angelo, F.; Leoni, L.; Stano, P. Gene-Expressing Liposomes as Synthetic Cells for Molecular Communication Studies. Front. Bioeng. Biotechnol. 2019,  DOI: 10.3389/fbioe.2019.00001
      There is no corresponding record for this reference.
    48. 48
      Frallicciardi, J.; Melcr, J.; Siginou, P.; Marrink, S. J.; Poolman, B. Membrane Thickness, Lipid Phase and Sterol Type Are Determining Factors in the Permeability of Membranes to Small Solutes. Nat. Commun. 2022, 13 (1), 1– 12,  DOI: 10.1038/s41467-022-29272-x
      There is no corresponding record for this reference.
    49. 49
      Díaz-álvarez, A. E.; Cadierno, V. Glycerol: A Promising Green Solvent and Reducing Agent for Metal-Catalyzed Transfer Hydrogenation Reactions and Nanoparticles Formation. Appl. Sci. 2013, 3 (1), 55– 69,  DOI: 10.3390/app3010055
      49
      Glycerol: a promising green solvent and reducing agent for metal-catalyzed transfer hydrogenation reactions and nanoparticles formation
      Diaz-Alvarez, Alba E.; Cadierno, Victorio
      Applied Sciences (2013), 3 (1), 55-69CODEN: ASPCC7; ISSN:2076-3417. (MDPI AG)
      Glycerol is a non-toxic, non-hazardous, non-volatile, biodegradable, and recyclable liq. that is generated as a byproduct in the manuf. of biodiesel fuel from vegetable oils. Due to its easy availability, along with its unique combination of phys. and chem. properties, glycerol has recently emerged as an economically appealing and safe solvent for org. synthesis. Recent works have also demonstrated that glycerol can be used as a hydrogen source in metal-catalyzed transfer hydrogenation of org. compds., such as aldehydes, ketones, olefins and nitroarenes. Herein, the advances reached in this emerging field are reviewed. The utility of glycerol as solvent and reducing agent for the generation of metal nanoparticles is also briefly discussed.
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    50. 50
      Azua, A.; Finn, M.; Yi, H.; Beatriz Dantas, A.; Voutchkova-Kostal, A. Transfer Hydrogenation from Glycerol: Activity and Recyclability of Iridium and Ruthenium Sulfonate-Functionalized N-Heterocyclic Carbene Catalysts. ACS Sustain. Chem. Eng. 2017, 5 (5), 3963– 3972,  DOI: 10.1021/acssuschemeng.6b03156
      There is no corresponding record for this reference.
    51. 51
      Śmigiel, W. M.; Lefrançois, P.; Poolman, B. Physicochemical Considerations for Bottom-up Synthetic Biology. Emerg. Top. Life Sci. 2019, 3 (5), 445– 458,  DOI: 10.1042/ETLS20190017
      51
      Physicochemical considerations for bottom-up synthetic biology
      Smigiel, Wojciech Mikolaj; Lefrancois, Pauline; Poolman, Bert
      Emerging Topics in Life Sciences (2019), 3 (5), 445-458CODEN: ETLSAG; ISSN:2397-8562. (Portland Press Ltd.)
      A review. The bottom-up construction of synthetic cells from mol. components is arguably one of the most challenging areas of research in the life sciences. We review the impact of confining biol. systems in synthetic vesicles. Complex cell-like systems require control of the internal pH, ionic strength, (macro)mol. crowding, redox state and metabolic energy conservation. These physicochem. parameters influence protein activity and need to be maintained within limits to ensure the system remains in steady-state. We present the physicochem. considerations for building synthetic cells with dimensions ranging from the smallest prokaryotes to eukaryotic cells.
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    52. 52
      Maloney, P. C.; Kashket, E. R.; Wilson, T. H. A Protonmotive Force Drives ATP Synthesis in Bacteria. Proc. Natl. Acad. Sci. U. S. A. 1974, 71 (10), 3896– 3900,  DOI: 10.1073/pnas.71.10.3896
      52
      Protonmotive force drives ATP synthesis in bacteria
      Maloney, Peter C.; Kashket, E. R.; Wilson, T. Hastings
      Proceedings of the National Academy of Sciences of the United States of America (1974), 71 (10), 3896-900CODEN: PNASA6; ISSN:0027-8424.
      When cells of Streptococcus lactis or Escherichia coli were suspended in a K-free medium, a membrane potential (neg. inside) could be artificially generated by the addn. of the K ionophore, valinomycin. In response to this inward directed protonmotive force, ATP synthesis catalyzed by the membrane-bound ATPase (EC 3.6.1.3) was obsd. The formation of ATP was not found in S. lactis that had been treated with the ATPase inhibitor, N,N'-dicyclohexylcarbodiimide, nor was it obsd. in a mutant of E. coli lacking the ATPase. Inhibition of ATP synthesis by S. lactis was also obsd. when the membrane potential was reduced by the presence of external K, or when cells were first incubated with the proton conductor, carbonyl cyanide fluoromethoxyphenylhydrazone. These results are in agreement with predictions made by the chemiosmotic hypothesis of Mitchell.
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    53. 53
      Strahl, H.; Hamoen, L. W. Membrane Potential Is Important for Bacterial Cell Division. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (27), 12281– 12286,  DOI: 10.1073/pnas.1005485107
      53
      Membrane potential is important for bacterial cell division
      Strahl, Henrik; Hamoen, Leendert W.
      Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (27), 12281-12286, S12281/1-S12281/12CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Many cell division-related proteins are located at specific positions in the bacterial cell, and this organized distribution of proteins requires energy. Here, the authors report that the proton motive force, or more specifically the (trans)membrane potential, is directly involved in protein localization. It emerged that the membrane potential modulates the distribution of several conserved cell division proteins such as MinD, FtsA, and the bacterial cytoskeletal protein MreB. The authors show for MinD that this is based on the membrane potential stimulated binding of its C-terminal amphipathic helix. This function of the membrane potential has implications for how these morphogenetic proteins work and provide an explanation for the effects obsd. with certain antimicrobial compds.
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    54. 54
      Bruice, T. C. A View at the Millennium: The Efficiency of Enzymatic Catalysis. Acc. Chem. Res. 2002, 35 (3), 139– 148,  DOI: 10.1021/ar0001665
      54
      A view at the millennium: the efficiency of enzymatic catalysis
      Bruice, Thomas C.
      Accounts of Chemical Research (2002), 35 (3), 139-148CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review and discussion with 58 refs. L. Pauling (1946) proposed that the active site of an enzyme (E) binds the transition state (TS) in preference to the substrate (S), and by doing so, stabilizes the TS and lowers the activation energy. E binding of TS in preference to S and increasing the TΔS⧧ by freezing out motions in E·S and E·TS have been accepted as the driving forces in enzymic catalysis. However, the smaller value of ΔG⧧ for a 1-substrate enzymic reaction, as compared to its nonenzymic counterpart, is generally the result of a smaller value of ΔH⧧. The TS in an enzymic reaction is reached through ground-state conformers that closely resemble the TS (near-attack conformers or NACs). E·NACs are in thermal equil. with all other E·S conformers and are turnstiles through which substrate mols. must pass to arrive at the lowest-energy TS. The TS in E·TS may or may not be bound tighter than the NAC is in E·NAC. Thus, the belief that enzymic reactions owe their facility to TS binding or to an increase in ΔStmo requires modification.
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    55. 55
      Feng, C.; Tollin, G.; Enemark, J. H. Sulfite Oxidizing Enzymes. Biochim. Biophys. Acta - Proteins Proteomics 2007, 1774 (5), 527– 539,  DOI: 10.1016/j.bbapap.2007.03.006
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      Sulfite oxidizing enzymes
      Feng, Changjian; Tollin, Gordon; Enemark, John H.
      Biochimica et Biophysica Acta, Proteins and Proteomics (2007), 1774 (5), 527-539CODEN: BBAPBW; ISSN:1570-9639. (Elsevier Ltd.)
      A review. Sulfite-oxidizing enzymes are essential mononuclear molybdoproteins involved in S metab. in animals, plants, and bacteria. There are 3 such enzymes presently known: (1) sulfite oxidase (SO) in animals, (2) SO in plants, and (3) sulfite dehydrogenase (SDH) in bacteria. X-ray crystal structures of enzymes from all 3 sources (chicken SO, Arabidopsis thaliana SO, and Starkeya novella SDH) show nearly identical square pyramidal coordination around the Mo atom, even though the overall structures of the proteins and the presence of addnl. cofactors vary. This structural information provides a mol. basis for studying the role of specific amino acids in catalysis. Animal SO catalyzes the final step in the degrdn. of S-contg. amino acids and is crit. in detoxifying excess sulfite. Human SO deficiency is a fatal genetic disorder that leads to early death, and impaired SO activity is implicated in sulfite neurotoxicity. Animal SO and bacterial SDH contain both Mo and heme domains, whereas plant SO only has the Mo domain. Intraprotein electron transfer (IET) between the Mo and Fe centers in animal SO and bacterial SDH is a key step in the catalysis, which can be studied by laser flash photolysis in the presence of deazariboflavin. IET studies on animal SO and bacterial SDH clearly demonstrate the similarities and differences between these 2 types of sulfite-oxidizing enzymes. Conformational change is involved in the IET of animal SO, in which electrostatic interactions may play a major role in guiding the docking of the heme domain to the Mo domain prior to electron transfer. In contrast, IET measurements for SDH demonstrate that IET occurs directly through the protein medium, which is distinctly different from that in animal SO. Point mutations in human SO can result in significantly impaired IET or no IET, thus rationalizing their fatal effects. The recent developments in the understanding of sulfite-oxidizing enzyme mechanisms that are driven by a combination of mol. biol., rapid kinetics, pulsed ESR, and computational techniques are the subject of this review.
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    56. 56
      Gong, W.; Hao, B.; Wei, Z.; Ferguson, D. J.; Tallant, T.; Krzycki, J. A.; Chan, M. K. Structure of the A2ε2 Ni-Dependent CO Dehydrogenase Component of the Methanosarcina Barkeri Acetyl-CoA Decarbonylase/Synthase Complex. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (28), 9558– 9563,  DOI: 10.1073/pnas.0800415105
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      Structure of the α2ε2 Ni-dependent CO dehydrogenase component of the Methanosarcina barkeri acetyl-CoA decarbonylase/synthase complex
      Gong, Weimin; Hao, Bing; Wei, Zhiyi; Ferguson, Donald J., Jr.; Tallant, Thomas; Krzycki, Joseph A.; Chan, Michael K.
      Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (28), 9558-9563CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Ni-dependent carbon monoxide dehydrogenases (Ni-CODHs) are a diverse family of enzymes that catalyze reversible CO:CO2 oxidoreductase activity in acetogens, methanogens, and some CO-using bacteria. Crystallog. of Ni-CODHs from CO-using bacteria and acetogens has revealed the overall fold of the Ni-CODH core and has suggested structures for the C cluster that mediates CO:CO2 interconversion. Despite these advances, the mechanism of CO oxidn. has remained elusive. Herein, we report the structure of a distinct class of Ni-CODH from methanogenic archaea: the α2ε2 component from the α8β8γ8δ8ε8 CODH/acetyl-CoA decarbonylase/synthase complex, an enzyme responsible for the majority of biogenic methane prodn. on Earth. The structure of this Ni-CODH component provides support for a hitherto unobserved state in which both CO and H2O/OH- bind to the Ni and the exogenous FCII iron of the C cluster, resp., and offers insight into the structures and functional roles of the ε-subunit and FeS domain not present in nonmethanogenic Ni-CODHs.
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    57. 57
      Wilcoxen, J.; Zhang, B.; Hille, R. Reaction of the Molybdenum- and Copper-Containing Carbon Monoxide Dehydrogenase from Oligotropha Carboxydovorans with Quinones. Biochemistry 2011, 50 (11), 1910– 1916,  DOI: 10.1021/bi1017182
      57
      Reaction of the Molybdenum- and Copper-Containing Carbon Monoxide Dehydrogenase from Oligotropha carboxydovorans with Quinones
      Wilcoxen, Jarett; Zhang, Bo; Hille, Russ
      Biochemistry (2011), 50 (11), 1910-1916CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Carbon monoxide dehydrogenase (CODH) from Oligotropha carboxydovorans catalyzes the oxidn. of carbon monoxide to carbon dioxide, providing the organism both a carbon source and energy for growth. In the oxidative half of the catalytic cycle, electrons gained from CO are ultimately passed to the electron transport chain of the Gram-neg. organism, but the proximal acceptor of reducing equiv. from the enzyme has not been established. Here we investigate the reaction of the reduced enzyme with various quinones and find them to be catalytically competent. Benzoquinone has a kox of 125.1 s-1 and a Kd of 48 μM. Ubiquinone-1 has a kox/Kd value of 2.88 × 105 M-1 s-1. 1,4-Naphthoquinone has a kox of 38 s-1 and a Kd of 140 μM, and 1,2-Naphthoquinone-4-sulfonic acid has a kox/Kd of 1.31 × 105 M-1 s-1. An extensive effort to identify a cytochrome that could be reduced by CO/CODH was unsuccessful. Steady-state studies with benzoquinone indicate that the rate-limiting step is in the reductive half of the reaction (i.e., the reaction of oxidized enzyme with CO). On the basis of the inhibition of CODH by diphenyliodonium chloride, we conclude that quinone substrates interact with CODH at the enzyme's flavin site. Our results strongly suggest that CODH donates reducing equiv. directly to the quinone pool without using a cytochrome as an intermediary.
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    58. 58
      Nishimura, H.; Nomura, Y.; Iwata, E.; Sato, N.; Sako, Y. Purification and Characterization of Carbon Monoxide Dehydrogenase from the Aerobic Hyperthermophilic Archaeon Aeropyrum Pernix. Fish. Sci. 2010, 76 (6), 999– 1006,  DOI: 10.1007/s12562-010-0277-8
      58
      Purification and characterization of carbon monoxide dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix
      Nishimura, Hiroshi; Nomura, Yoshiko; Iwata, Eri; Sato, Nozomi; Sako, Yoshihiko
      Fisheries Science (Tokyo, Japan) (2010), 76 (6), 999-1006CODEN: FSCIEH; ISSN:0919-9268. (Springer Japan)
      The aerobic hyperthermophilic archaeon Aeropyrum pernix expresses carbon monoxide (CO) oxidn. activity under heterotrophic growth conditions. Using activity stain gel anal., CO oxidn. activity was detected in a protein with a mol. mass of 210 kDa. The 210 kDa CODH protein was purified to homogeneity from A. pernix. Aeropyrum Mo-CODH catalyzed the oxidn. of CO with a specific activity of 2.1 μmol CO min-1 mg-1 at 95°C, pH 8.0 using Me viologen as the electron acceptor. The CODH protein showed high oxygen and thermo stability. The protein contains three subunits: L (86.6 kDa), M (34.5 kDa), and S (12.6 kDa), which form the LM2S complex. The mol. mass of the complex was calcd. by gel filtration and found to be 163.7 kDa. N-terminal amino acid sequencing and peptide mass fingerprinting anal. of the subunits indicated that they corresponded to NP_148462.1, NP_148464.2, and NP_148465.1, and their genes annotated the molybdo iron-sulfur flavoprotein carbon monoxide dehydrogenase S, L, and M subunits, resp. Phylogenetic anal. revealed that CODH belongs to a novel clade of diverse CODHs.
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    59. 59
      Ruzheinikov, S. N.; Burke, J.; Sedelnikova, S.; Baker, P. J.; Taylor, R.; Bullough, P. A.; Muir, N. M.; Gore, M. G.; Rice, D. W. Glycerol Dehydrogenase: Structure, Specificity, and Mechanism of a Family III Polyol Dehydrogenase. Structure 2001, 9 (9), 789– 802,  DOI: 10.1016/S0969-2126(01)00645-1
      59
      Glycerol dehydrogenase structure, specificity, and mechanism of a family III polyol dehydrogenase
      Ruzheinikov, S. N.; Burke, J.; Sedelnikova, S.; Baker, P. J.; Taylor, R.; Bullough, P. A.; Muir, N. M.; Gore, M. G.; Rice, D. W.
      Structure (Cambridge, MA, United States) (2001), 9 (9), 789-802CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
      Bacillus stearothermophilus glycerol dehydrogenase (EC 1.1.1.6) (I) catalyzes the oxidn. of glycerol to dihydroxyacetone with the concomitant redn. of NAD to NADH. Anal. of the sequence of this enzyme indicates that it is a member of the so-called Fe-contg. alc. dehydrogenase family. Despite this sequence similarity, I shows a strict dependence on Zn for activity. On the basis of this, the authors propose to rename this group the family III metal-dependent polyol dehydrogenases. To date, no structural data have been reported for any enzyme in this group. Here, the crystal structure of B. stearothermophilus I was detd. at 1.7 Å resoln. to provide structural insights into the mechanistic features of this family. I has 370 amino acid residues, has a mol. wt. of 39.5 kDa, and is a homooctamer in soln. Anal. of the crystal structures of the free enzyme and of its binary complexes with NAD and glycerol showed that the active site of I was in the cleft between the enzyme's 2 domains, with the catalytic Zn2+ ion playing a role in stabilizing an alkoxide intermediate. In addn., the specificity of I for a range of diols could be understood, as both OH groups of glycerol formed ligands to the enzyme-bound Zn2+ ion at the active site. The structure further revealed a previously unsuspected similarity to dehydroquinate synthase, an enzyme whose more complex chem. shares a common chem. step with that catalyzed by I, providing a striking example of divergent evolution. Finally, the structure suggested that the NAD-binding domain of I may be related to that of the classical Rossmann fold by switching the sequence order of the 2 mononucleotide binding folds that make up this domain.
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    60. 60
      Heck, d. H. A.; Casanova, M.; Starr, T. B. Formaldehyde Toxicity - New Understanding. Crit. Rev. Toxicol. 1990, 20 (6), 397– 426,  DOI: 10.3109/10408449009029329
      60
      Formaldehyde toxicity. New understanding
      Heck, Henry d'A.; Casanova, Mercedes; Starr, Thomas B.
      Critical Reviews in Toxicology (1990), 20 (6), 397-426CODEN: CRTXB2; ISSN:0045-6446.
      A review with 235 refs. on metab., toxicity, mutagenicity, reactions with macromols., mol. dosimetry, epidemiol., and risk assessment of formaldehyde.
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    61. 61
      Muñoz-Elías, E. J.; McKinney, J. D. Carbon Metabolism of Intracellular Bacteria. Cell. Microbiol. 2006, 8 (1), 10– 22,  DOI: 10.1111/j.1462-5822.2005.00648.x
      61
      Carbon metabolism of intracellular bacteria
      Munoz-Elias, Ernesto J.; McKinney, John D.
      Cellular Microbiology (2006), 8 (1), 10-22CODEN: CEMIF5; ISSN:1462-5814. (Blackwell Publishing Ltd.)
      A review. Bacterial metab. was studied intensively since the 1st observations of these 'animalcules' by Leeuwenhoek and their isolation in pure cultures by Pasteur. Metabolic studies have traditionally focused on a small no. of model organisms, primarily the Gram neg. bacillus Escherichia coli, adapted to artificial culture conditions in the lab. Comparatively little is known about the physiol. and metab. of wild microorganisms living in their natural habitats. For ∼500-1000 species of commensals and symbionts, and a smaller no. of pathogenic bacteria, that habitat is the human body. Emerging evidence suggests that the metab. of bacteria grown in vivo differs profoundly from their metab. in axenic cultures.
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    62. 62
      Hochstein, L. I.; Tomlinson, G. A. The Enzymes Associated with Denitrification. Annu. Rev. Microbiol. 1988, 42 (72), 231– 261,  DOI: 10.1146/annurev.mi.42.100188.001311
      62
      The enzymes associated with denitrification
      Hochstein, Lawrence I.; Tomlinson, Geraldine A.
      Annual Review of Microbiology (1988), 42 (), 231-61CODEN: ARMIAZ; ISSN:0066-4227.
      A review with 170 refs. on the enzyme systems which confer upon some eubacteria and archaebacteria the ability to grow anaerobically by reducing ionic nitrogenous oxides to gaseous products. The topics discussed include nitrate reductase, nitrite reductases, nitric oxide reductases, mechanism of N-N bond formation, and nitrous oxide reductases.
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    63. 63
      Dolla, A.; Fournier, M.; Dermoun, Z. Oxygen Defense in Sulfate-Reducing Bacteria. J. Biotechnol. 2006, 126 (1), 87– 100,  DOI: 10.1016/j.jbiotec.2006.03.041
      63
      Oxygen defense in sulfate-reducing bacteria
      Dolla, Alain; Fournier, Marjorie; Dermoun, Zorah
      Journal of Biotechnology (2006), 126 (1), 87-100CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)
      A review. Sulfate-reducing bacteria (SRB) are strict anaerobes that are often found in biotopes where oxic conditions can temporarily exist. The bacteria have developed several defense strategies to survive exposure to oxygen. These strategies includes peculiar behaviors in the presence of oxygen, like aggregation or aerotaxis, and enzymic systems dedicated to the redn. and the elimination of oxygen and its reactive species. Sulfate-reducing bacteria, and specially Desulfovibrio species, possess a variety of enzymes acting together to achieve an efficient defense against oxidative stress. The function and occurrence of these enzymic systems are described.
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    64. 64
      Lu, Z.; Imlay, J. A. When Anaerobes Encounter Oxygen: Mechanisms of Oxygen Toxicity, Tolerance and Defence. Nat. Rev. Microbiol. 2021, 19 (12), 774– 785,  DOI: 10.1038/s41579-021-00583-y
      64
      When anaerobes encounter oxygen: mechanisms of oxygen toxicity, tolerance and defence
      Lu, Zheng; Imlay, James A.
      Nature Reviews Microbiology (2021), 19 (12), 774-785CODEN: NRMACK; ISSN:1740-1526. (Nature Portfolio)
      Abstr.: The defining trait of obligate anaerobes is that oxygen blocks their growth, yet the underlying mechanisms are unclear. A popular hypothesis was that these microorganisms failed to evolve defences to protect themselves from reactive oxygen species (ROS) such as superoxide and hydrogen peroxide, and that this failure is what prevents their expansion to oxic habitats. However, studies reveal that anaerobes actually wield most of the same defences that aerobes possess, and many of them have the capacity to tolerate substantial levels of oxygen. Therefore, to understand the structures and real-world dynamics of microbial communities, investigators have examd. how anaerobes such as Bacteroides, Desulfovibrio, Pyrococcus and Clostridium spp. struggle and cope with oxygen. The hypoxic environments in which these organisms dwell - including the mammalian gut, sulfur vents and deep sediments - experience episodic oxygenation. In this Review, we explore the mol. mechanisms by which oxygen impairs anaerobes and the degree to which bacteria protect their metabolic pathways from it. The emergent view of anaerobiosis is that optimal strategies of anaerobic metab. depend upon radical chem. and low-potential metal centers. Such catalytic sites are intrinsically vulnerable to direct poisoning by mol. oxygen and ROS. Observations suggest that anaerobes have evolved tactics that either minimize the extent to which oxygen disrupts their metab. or restore function shortly after the stress has dissipated.
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    65. 65
      Patil, Y. P.; Jadhav, S. Novel Methods for Liposome Preparation. Chem. Phys. Lipids 2014, 177, 8– 18,  DOI: 10.1016/j.chemphyslip.2013.10.011
      65
      Novel methods for liposome preparation
      Patil, Yogita P.; Jadhav, Sameer
      Chemistry and Physics of Lipids (2014), 177 (), 8-18CODEN: CPLIA4; ISSN:0009-3084. (Elsevier Ltd.)
      A review. Liposomes are bilayer vesicles which have found use, among other applications, as drug delivery vehicles. Conventional techniques for liposome prepn. and size redn. remain popular as these are simple to implement and do not require sophisticated equipment. However, issues related to scale-up for industrial prodn. and scale-down for point-of-care applications have motivated improvements to conventional processes and have also led to the development of novel routes to liposome formation. In this article, these modified and new methods for liposome prepn. have been reviewed and classified with the objective of updating the reader to recent developments in liposome prodn. technol.
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    66. 66
      Mitchell, P. Coupling of Phosphorylation to Electron and Hydrogen Transfer by a Chemi-Osmotic Type of Mechanism. Nature 1961, 191 (4784), 144– 148,  DOI: 10.1038/191144a0
      66
      Coupling of phosphorylation to electron and hydrogen transfer by a chemiosmotic type of mechanism
      Mitchell, Peter
      Nature (London, United Kingdom) (1961), 191 (), 144-8CODEN: NATUAS; ISSN:0028-0836.
      A chemiosmotic mechanism dependent on the supramol. organization (membrane structure) of multienzyme systems is proposed in contrast to the orthodox substrate-enzyme type of coupling. The driving force is postulated as due to spatially directed diffusion of the active components. An essential requirement is the presence within an ion-impermeable membrane of an anisotropic reversible adenosine triphosphatase (ATPase) system (active center). In such an active center (e.g. phosphokinase), the hydrolysis equil. in the adenosine triphosphate (ATP)-adenosine diphosphate (ADP) system is detd. by the electrochem. activity of the H2O at the active center, [H2O]c = [H+]R[OH]L = [H+]R[H2O]aq./[H+]L where R (right) and L (left) designate the cytoplasmic and intracellular aq. phases, resp. The electrochem. activity ratio for the enzyme system, including the elements of H2O, is given as: [ATP]/[ADP] = {[P]/K[H2O]} {[H+]L/[H+]R}. At pH 7, K1[H2O]aq. ∼105; where [P] is at physiol. levels (approx. 10-2M), the ratio is: [ATP]/[ADP] equal or nearly equal to {[H+]L/[H+]R} × 10-7. Reversal of enzyme activity is kinetically explained on the basis of the electrochem. activity gradient of H+ and OH- across the active center. The fundamental processes essentially involve dehydroxylation and deprotonation. The [H+] and [OH-] gradients are governed by an anisotropic electron-chain transfer (reoxidn.-redn.) mechanism coupled to the reversible ATPase system of the active center. One ATP mol. is produced per electron transfer when the oxidn.-redn. and phosphorylation systems are in chemiosmotic equil. The energy relation is expressed as [ATP]/[ADP] = {[P]/105} × 10ΔE/60, where ΔE (the oxidn.-redn. span) is equiv. to the free energy change in mv./electron transferred. At inorg. phosphate concns. of 10-2M this would require a min. ΔE of 420 mv. to drive the ATP synthesis. Such energies are readily available through the coenzyme (nucleotides, flavoproteins, ubiquinones) and the carboxylic acid (succinic-fumarate) systems. Diagrammatic representations are shown for the coupling systems. Loose membrane structures (or equiv. uncoupling agents like dinitrophenols) are postulated as disrupting the chemiosmotic coupling mechanism with consequent change in the energy relations of the steady state. The chemiosmotic hypothesis is utilized to explain photophosphorylation and also a number of other facts (absence of energy-rich intermediates; dependence of coupling on membrane structure and its ion impermeability; differential effects of [H+]; action of uncoupling agents; membrane swelling and shrinkage) difficult to reconcile on the basis of classical concepts. In some concluding speculations, membrane transport and metabolism are considered as simply different aspects of a unifying process, termed vectorial metabolism.
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    67. 67
      Russell, J. B. The Energy Spilling Reactions of Bacteria and Other Organisms. J. Mol. Microbiol. Biotechnol. 2007, 13 (1–3), 1– 11,  DOI: 10.1159/000103591
      67
      The energy spilling reactions of bacteria and other organisms
      Russell, James B.
      Journal of Molecular Microbiology and Biotechnology (2007), 13 (1-3), 1-11CODEN: JMMBFF; ISSN:1464-1801. (S. Karger AG)
      A review. For many years, it was assumed that living organisms always utilized ATP in a highly efficient manner, but simple growth studies with bacteria indicated that the efficiency of biomass prodn. was often at least 3-fold lower than the amt. that would be predicted from std. biosynthetic pathways. The utilization of energy for maintenance could only explain a small portion of this discrepancy particularly when the growth rate was high. These ideas and thermodn. arguments indicated that cells might have another avenue of energy utilization. This phenomenon has also been called 'uncoupling', 'spillage' and 'overflow metab.', but 'energy spilling' is probably the most descriptive term. It appears that many bacteria spill energy, and the few that do not can be killed (large and often rapid decrease in viability), if the growth medium is nitrogen-limited and the energy source is in 'excess'. The lactic acid bacterium, Streptococcus bovis, is an ideal bacterium for the study of energy spilling. Because it only uses substrate level phosphorylation to generate ATP, ATP generation can be calcd. with a high degree of certainty. It does not store glucose as glycogen, and its cell membrane can be easily accessed. Comparative anal. of heat prodn., membrane voltage, ATP prodn. and Ohm's law indicated that the energy spilling reaction of S. bovis is mediated by a futile cycle of protons through the cell membrane. Less is known about Escherichia coli, but in this bacterium energy spilling could be mediated by a futile cycle of potassium or ammonium ions. Energy spilling is not restricted to prokaryotes and appears to occur in yeasts and in higher organisms. In man, energy spilling may be related to cancer, aging, ischemia and cardiac failure.
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    68. 68
      Jin, Q. Energy Conservation of Anaerobic Respiration. Am. J. Sci. 2012, 312 (6), 573– 628,  DOI: 10.2475/06.2012.01
      68
      Energy conservation of anaerobic respiration
      Jin, Qusheng
      American Journal of Science (2012), 312 (6), 573-628CODEN: AJSCAP; ISSN:0002-9599. (Kline Geology Laboratory)
      A review. Microbes save energy from the environment by synthesizing ATP. Understanding microbial energy conservation is important for both theor. and practical applications. This paper focuses on the common metab. of anoxic environments-ferric iron respiration, sulfate respiration, and methanogenesis-and analyzes microbial energy conservation on the basis of the thermodn. as well as physiol. models of respiratory reactions. The results of the anal. show that iron respiration synthesizes 1 to 4 ATPs by transferring eight electrons from H2, acetate, lactate, and ethanol to ferric minerals; sulfate respiration makes 0.25 to more than 3 ATPs by transferring eight electrons from the same suite of electron donors to sulfate; methanogenesis yields 0 to 1 ATP by oxidizing four H2 and by disproportionating one acetate. The ATP yields are compared to growth yields and energy thresholds of anaerobic metab. to explore the impact of energy conservation. Specifically, energy conservation controls microbial growth: in geochem. systems, respiring microbes synthesize up to 5 g biomass per mol of ATP. Energy conservation also requires the environment to supply chem. energy at quantities greater than the energy saved by microbes. These results unify our view of microbial metab., and can be applied to evaluating the occurrence and significance of microbial life in natural environments.
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    69. 69
      Jones, A. J. Y.; Blaza, J. N.; Bridges, H. R.; May, B.; Moore, A. L.; Hirst, J. A Self-Assembled Respiratory Chain That Catalyzes NADH Oxidation by Ubiquinone-10 Cycling between ComplexI and the Alternative Oxidase. Angew. Chemie - Int. Ed. 2016, 55 (2), 728– 731,  DOI: 10.1002/anie.201507332
      69
      A Self-Assembled Respiratory Chain that Catalyzes NADH Oxidation by Ubiquinone-10 Cycling between Complex I and the Alternative Oxidase
      Jones, Andrew J. Y.; Blaza, James N.; Bridges, Hannah R.; May, Benjamin; Moore, Anthony L.; Hirst, Judy
      Angewandte Chemie, International Edition (2016), 55 (2), 728-731CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Complex I is a crucial respiratory enzyme that conserves the energy from NADH oxidn. by ubiquinone-10 (Q10) in proton transport across a membrane. Studies of its energy transduction mechanism are hindered by the extreme hydrophobicity of Q10, and they have so far relied on native membranes with many components or on hydrophilic Q10 analogs that partition into membranes and undergo side reactions. Herein, we present a self-assembled system without these limitations: proteoliposomes contg. mammalian complex I, Q10, and a quinol oxidase (the alternative oxidase, AOX) to recycle Q10H2 to Q10. AOX is present in excess, so complex I is completely rate detg. and the Q10 pool is kept oxidized under steady-state catalysis. The system was used to measure a fully-defined KM value for Q10. The strategy is suitable for any enzyme with a hydrophobic quinone/quinol substrate, and could be used to characterize hydrophobic inhibitors with potential applications as pharmaceuticals, pesticides, or fungicides.
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    70. 70
      Biner, O.; Fedor, J. G.; Yin, Z.; Hirst, J. Bottom-Up Construction of a Minimal System for Cellular Respiration and Energy Regeneration. ACS Synth. Biol. 2020, 9 (6), 1450– 1459,  DOI: 10.1021/acssynbio.0c00110
      70
      Bottom-Up Construction of a Minimal System for Cellular Respiration and Energy Regeneration
      Biner, Olivier; Fedor, Justin G.; Yin, Zhan; Hirst, Judy
      ACS Synthetic Biology (2020), 9 (6), 1450-1459CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      ATP, the cellular energy currency, is essential for life. The ability to provide a const. supply of ATP is therefore crucial for the construction of artificial cells in synthetic biol. Here, the authors describe the bottom-up assembly and characterization of a minimal respiratory system that uses NADH as a fuel to produce ATP from ADP and inorg. phosphate, and is thus capable of sustaining both upstream metabolic processes that rely on NAD+, and downstream energy-demanding processes that are powered by ATP hydrolysis. A detergent-mediated approach was used to coreconstitute respiratory mitochondrial complex I and an F-type ATP synthase into nanosized liposomes. Addn. of the alternative oxidase to the resulting proteoliposomes produced a minimal artificial "organelle" that reproduces the energy-converting catalytic reactions of the mitochondrial respiratory chain: NADH oxidn., ubiquinone cycling, oxygen redn., proton pumping, and ATP synthesis. As a proof-of-principle, the authors demonstrate that the nanovesicles are capable of using an NAD+-linked substrate to drive cell-free protein expression. The nanovesicles are both efficient and durable and may be applied to sustain artificial cells in future work.
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    71. 71
      Norris, F. A.; Powell, G. L. The Apparent Permeability Coefficient for Proton Flux through Phosphatidylcholine Vesicles Is Dependent on the Direction of Flux. BBA - Biomembr. 1990, 1030 (1), 165– 171,  DOI: 10.1016/0005-2736(90)90252-J
      There is no corresponding record for this reference.
    72. 72
      Garcia Costas, A. M.; White, A. K.; Metcalf, W. W. Purification and Characterization of a Novel Phosphorus-Oxidizing Enzyme from Pseudomonas Stutzeri WM88. J. Biol. Chem. 2001, 276 (20), 17429– 17436,  DOI: 10.1074/jbc.M011764200
      72
      Purification and characterization of a novel phosphorus-oxidizing enzyme from Pseudomonas stutzeri WM88
      Garcia Costas, Amaya M.; White, Andrea K.; Metcalf, William W.
      Journal of Biological Chemistry (2001), 276 (20), 17429-17436CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The ptxD gene from Pseudomonas stutzeri WM88 encoding the novel phosphorus oxidizing enzyme NAD:phosphite oxidoreductase (trivial name phosphite dehydrogenase, PtxD) was cloned into an expression vector and over-produced in Escherichia coli. The heterologously produced enzyme is indistinguishable from the native enzyme based on mass spectrometry, amino-terminal sequencing, and specific activity analyses. Recombinant PtxD was purified to homogeneity via a two-step affinity protocol and characterized. The enzyme stoichiometrically produces NADH and phosphate from NAD and phosphite. The reverse reaction was not obsd. Gel filtration anal. of the purified protein is consistent with PtxD acting as a homodimer. PtxD has a high affinity for its substrates with Km values of 53.1 ± 6.7 μM and 54.6 ± 6.7 μM, for phosphite and NAD, resp. Vmax and kcat were detd. to be 12.2 ± 0.3 μmol min-1 mg-1 and 440 min-1. NADP can substitute poorly for NAD; however, none of the numerous compds. examd. were able to substitute for phosphite. Initial rate studies in the absence or presence of products and in the presence of the dead end inhibitor sulfite are most consistent with a sequential ordered mechanism for the PtxD reaction, with NAD binding first and NADH being released last. Amino acid sequence comparisons place PtxD as a new member of the D-2-hydroxyacid NAD-dependent dehydrogenases, the only one to have an inorg. substrate. To our knowledge, this is the first detailed biochem. study on an enzyme capable of direct oxidn. of a reduced phosphorus compd.
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    73. 73
      Popov, V. O.; Lamzin, V. S. NAD+-Dependent Formate Dehydrogenase. Biochem. J. 1994, 301 (3), 625,  DOI: 10.1042/bj3010625
      73
      NAD+-dependent formate dehydrogenase
      Popov, Vladimir O.; Lamzin, Victor S.
      Biochemical Journal (1994), 301 (3), 625-43CODEN: BIJOAK; ISSN:0264-6021.
      A review with 158 refs., of the kinetic properties and structure of the title enzyme.
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    74. 74
      Tishkov, V. I.; Popov, V. O. Catalytic Mechanism and Application of Formate Dehydrogenase. Biochem. 2004, 69 (11), 1252– 1267,  DOI: 10.1007/s10541-005-0071-x
      74
      Catalytic mechanism and application of formate dehydrogenase
      Tishkov, V. I.; Popov, V. O.
      Biochemistry (Moscow, Russian Federation) (2004), 69 (11), 1252-1267, 1 plateCODEN: BIORAK; ISSN:0006-2979. (MAIK Nauka/Interperiodica Publishing)
      A review. NAD-dependent formate dehydrogenase (FDH) is an abundant enzyme that plays an important role in energy supply of methylotrophic microorganisms and in response to stress in plants. FDH belongs to the superfamily of D-specific 2-hydroxy acid dehydrogenases. FDH is widely accepted as a model enzyme to study the mechanism of hydride ion transfer in the active center of dehydrogenases because the reaction catalyzed by the enzyme is devoid of proton transfer steps and implies a substrate with relatively simple structure. FDH is also widely used in enzymic syntheses of optically active compds. as a versatile biocatalyst for NAD(P)H regeneration consumed in the main reaction. This review covers recent developments in cloning genes of FDH from various sources, studies of its catalytic mechanism and physiol. role, and its application for new chiral syntheses.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtVWjs7g%253D&md5=258e2ff8424c8ca05d84197ca4680453
    75. 75
      Schneider, K.; Schlegel, H. G. Purification and Properties of Soluble Hydrogenase from Alcaligenes Eutrophus H 16. Biochim. Biophys. Acta (BBA)-Enzymology 1976, 452 (1), 66– 80,  DOI: 10.1016/0005-2744(76)90058-9
      There is no corresponding record for this reference.
    76. 76
      Burgdorf, T.; van der Linden, E.; Bernhard, M.; Yin, Q. Y.; Back, J. W.; Hartog, A. F.; Muijsers, A. O.; de Koster, C. G.; Albracht, S. P. J.; Friedrich, B. The Soluble NAD+-Reducing [NiFe]-Hydrogenase from Ralstonia Eutropha H16 Consists of Six Subunits and Can Be Specifically Activated by NADPH. J. Bacteriol. 2005, 187 (9), 3122– 3132,  DOI: 10.1128/JB.187.9.3122-3132.2005
      76
      The soluble NAD+-reducing [NiFe]-hydrogenase from Ralstonia eutropha H16 consists of six subunits and can be specifically activated by NADPH
      Burgdorf, Tanja; van der Linden, Eddy; Bernhard, Michael; Yin, Qing Yuan; Back, Jaap W.; Hartog, Aloysius F.; Muijsers, Anton O.; de Koster, Chris G.; Albracht, Simon P. J.; Friedrich, Baerbel
      Journal of Bacteriology (2005), 187 (9), 3122-3132CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      The sol. [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates redn. of NAD+ at the expense of H2. We have purified a new high-mol.-wt. form of the SH which contains an addnl. subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degrdn., in combination with protein sequencing of the SH high-mol.-wt. complex, established a subunit stoichiometry of HoxFUYHI2. Crosslinking expts. indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amts. of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amts. of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex I).
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    77. 77
      Herr, N.; Ratzka, J.; Lauterbach, L.; Lenz, O.; Ansorge-Schumacher, M. B. Stability Enhancement of an O2-Tolerant NAD+-Reducing [NiFe]-Hydrogenase by a Combination of Immobilisation and Chemical Modification. J. Mol. Catal. B Enzym. 2013, 97, 169– 174,  DOI: 10.1016/j.molcatb.2013.06.009
      77
      Stability enhancement of an O2-tolerant NAD+-reducing [NiFe|-hydrogenase by a combination of immobilization and chemical modification
      Herr, Nicole; Ratzka, Juliane; Lauterbach, Lars; Lenz, Oliver; Ansorge-Schumacher, Marion B.
      Journal of Molecular Catalysis B: Enzymatic (2013), 97 (), 169-174CODEN: JMCEF8; ISSN:1381-1177. (Elsevier B.V.)
      The oxygen-tolerant, NAD+-reducing sol. hydrogenase (SH) from Ralstonia eutropha H16 is a promising catalyst for cofactor regeneration in enzyme-catalyzed redn. processes. The tech. use of the isolated enzyme, however, is limited by its relatively low stability under operational conditions such as agitation, elevated temp. or addn. of co-solvents. The max. half-life at a reaction temp. of 35° and pH 8.0 was only 5.3 h. In order to enhance the stability of the enzyme, it was immobilized onto the anionic resin Amberlite FPA54. At an immobilization yield of 93.4% for adsorptive and 100% for covalent attachment, corresponding activities of 48.9 and 39.3%, resp., were obtained. Covalent binding always yielded superior stabilization. At elevated temp. and under agitation, stabilization was further increased by modification of the covalently bound SH with methoxy-poly(ethylene) glycol (mPEG). A comparable effect was not achieved when SH modification was performed before immobilization. In stationary aq. soln., half-lives of up to 161 h at 25° and 32 h at 35° were obtained. In presence of the tech. relevant co-solvents DMSO, DMF, 2-propanol and [EMIM][EtSO4] half-lives of 14-29 h can now be achieved.
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    78. 78
      Velasco-Lozano, S.; Roca, M.; Leal-Duaso, A.; Mayoral, J. A.; Pires, E.; Moliner, V.; López-Gallego, F. Selective Oxidation of Alkyl and Aryl Glyceryl Monoethers Catalysed by an Engineered and Immobilised Glycerol Dehydrogenase. Chem. Sci. 2020, 11 (44), 12009– 12020,  DOI: 10.1039/D0SC04471G
      78
      Selective oxidation of alkyl and aryl glyceryl monoethers catalysed by an engineered and immobilized glycerol dehydrogenase
      Velasco-Lozano, Susana; Roca, Maite; Leal-Duaso, Alejandro; Mayoral, Jose A.; Pires, Elisabet; Moliner, Vicent; Lopez-Gallego, Fernando
      Chemical Science (2020), 11 (44), 12009-12020CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Enzymes acting over glyceryl ethers are scarce in living cells, and consequently biocatalytic transformations of these mols. are rare despite their interest for industrial chem. In this work, we have engineered and immobilized a glycerol dehydrogenase from Bacillus stearothermophilus (BsGlyDH) to accept a battery of alkyl/aryl glyceryl monoethers and catalyze their enantioselective oxidn. to yield the corresponding 3-alkoxy/aryloxy-1-hydroxyacetones. QM/MM computational studies decipher the key role of D123 in the oxidn. catalytic mechanism, and reveal that this enzyme is highly enantioselective towards S-isomers (ee > 99%). Through structure-guided site-selective mutagenesis, we find that the mutation L252A sculpts the active site to accommodate a productive configuration of 3-monoalkyl glycerols. This mutation enhances the kcat 163-fold towards 3-ethoxypropan-1,2-diol, resulting in a specific activity similar to the one found for the wild-type towards glycerol. Furthermore, we immobilized the L252A variant to intensify the process, demonstrating the reusability and increasing the operational stability of the resulting heterogeneous biocatalyst. Finally, we manage to integrate this immobilized enzyme into a one-pot chemoenzymic process to convert glycidol and ethanol into 3-ethoxy-1-hydroxyacetone and (R)-3-ethoxypropan-1,2-diol, without affecting the oxidn. activity. These results thus expand the uses of engineered glycerol dehydrogenases in applied biocatalysis for the kinetic resoln. of glycerol ethers and the manufg. of substituted hydroxyacetones.
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    79. 79
      Matelska, D.; Shabalin, I. G.; Jabłońska, J.; Domagalski, M. J.; Kutner, J.; Ginalski, K.; Minor, W. Classification, Substrate Specificity and Structural Features of D-2-Hydroxyacid Dehydrogenases: 2HADH Knowledgebase. BMC Evol. Biol. 2018, 18 (1), 1– 23,  DOI: 10.1186/s12862-018-1309-8
      79
      Classification, substrate specificity and structural features of D-2-hydroxyacid dehydrogenases: 2HADH knowledgebase
      Matelska, Dorota; Shabalin, Ivan G.; Jablonska, Jagoda; Domagalski, Marcin J.; Kutner, Jan; Ginalski, Krzysztof; Minor, Wladek
      BMC Evolutionary Biology (2018), 18 (1), 1-23CODEN: BEBMCG; ISSN:1471-2148. (BioMed Central Ltd.)
      We report an in-depth phylogenetic anal., followed by mapping of available biochem. and structural data on the reconstructed phylogenetic tree. The anal. suggests that some subfamilies comprising enzymes with similar yet broad substrate specificity profiles diverged early in the evolution of 2HADHs. Based on the phylogenetic tree, we present a revised classification of the family that comprises 22 subfamilies, including 13 new subfamilies not studied biochem. We summarize characteristics of the nine biochem. studied subfamilies by aggregating all available sequence, biochem., and structural data, providing comprehensive descriptions of the active site, cofactor-binding residues, and potential roles of specific structural regions in substrate recognition. In addn., we concisely present our anal. as an online 2HADH enzymes knowledgebase. Conclusions: The knowledgebase enables navigation over the 2HADHs classification, search through collected data, and functional predictions of uncharacterized 2HADHs. Future characterization of the new subfamilies may result in discoveries of enzymes with novel metabolic roles and with properties beneficial for biotechnol. applications.
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    80. 80
      Woodyer, R.; Van der Donk, W. A.; Zhao, H. Relaxing the Nicotinamide Cofactor Specificity of Phosphite Dehydrogenase by Rational Design. Biochemistry 2003, 42 (40), 11604– 11614,  DOI: 10.1021/bi035018b
      80
      Relaxing the nicotinamide cofactor specificity of phosphite dehydrogenase by rational design
      Woodyer, Ryan; van der Donk, Wilfred A.; Zhao, Huimin
      Biochemistry (2003), 42 (40), 11604-11614CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Homol. modeling was used to identify two particular residues, Glu175 and Ala176, in Pseudomonas stutzeri phosphite dehydrogenase(PTDH) as the principal determinants of nicotinamide cofactor (NAD+ and NADP+) specificity. Replacement of these two residues by site-directed mutagenesis with Ala175 and Arg176 both sep. and in combination resulted in PTDH mutants with relaxed cofactor specificity. All three mutants exhibited significantly better catalytic efficiency for both cofactors, with the best kinetic parameters displayed by the double mutant, which had a 3.6-fold higher catalytic efficiency for NAD+ and a 1000-fold higher efficiency for NADP+. The cofactor specificity was changed from 100-fold in favor of NAD+ for the wild-type enzyme to 3-fold in favor of NADP+ for the double mutant. Isoelec. focusing of the proteins in a nondenaturing gel showed that the replacement with more basic residues indeed changed the effective pI of the protein. HPLC anal. of the enzymic products of the double mutant verified that the reaction proceeded to completion using either substrate and produced only the corresponding reduced cofactor and phosphate. Thermal inactivation studies showed that the double mutant was protected from thermal inactivation by both cofactors, while the wild-type enzyme was protected by only NAD+. The combined results provide clear evidence that Glu175 and Ala176 are both crit. for nicotinamide cofactor specificity. The rationally designed double mutant might be useful for the development of an efficient in vitro NAD(P)H regeneration system for reductive biocatalysis.
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    81. 81
      Metcalf, W. W.; Wolfe, R. S. Molecular Genetic Analysis of Phosphite and Hypophosphite Oxidation by Pseudomonas Stutzeri WM88. J. Bacteriol. 1998, 180 (21), 5547– 5558,  DOI: 10.1128/JB.180.21.5547-5558.1998
      81
      Molecular genetic analysis of phosphite and hypophosphite oxidation by Pseudomonas stutzeri WM88
      Metcalf, William W.; Wolfe, Ralph S.
      Journal of Bacteriology (1998), 180 (21), 5547-5558CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      The first mol. and genetic characterization of a biochem. pathway for oxidn. of the reduced phosphorus (P) compds. phosphite and hypophosphite is reported. The pathway was identified in Pseudomonas stutzeri WM88, which was chosen for detailed studies from a group of organisms isolated based on their ability to oxidize hypophosphite (+1 valence) and phosphite (+3 valence) to phosphate (+5 valence). The genes required for oxidn. of both compds. by P. stutzeri WM88 were cloned on a single ca. 30-kbp DNA fragment by screening for expression in Escherichia coli and Pseudomonas aeruginosa. Two lines of evidence suggest that hypophosphite is oxidized to phosphate via a phosphite intermediate. First, plasmid subclones that conferred oxidn. of phosphite, but not hypophosphite, upon heterologous hosts were readily obtained. All plasmid subclones that failed to confer phosphite oxidn. also failed to confer hypophosphite oxidn. No subclones that conferred only hypophosphite expression were obtained. Second, various deletion derivs. of the cloned genes were made in vitro and recombined onto the chromosome of P. stutzeri WM88. Two phenotypes were displayed by individual mutants. Mutants with the region encoding phosphite oxidn. deleted (based upon the subcloning results) lost the ability to oxidize either phosphite or hypophosphite. Mutants with the region encoding hypophosphite oxidn. deleted lost only the ability to oxidize hypophosphite. The phenotypes displayed by these mutants also demonstrate that the cloned genes are responsible for the P oxidn. phenotypes displayed by the original P. stutzeri WM88 isolate. The DNA sequences of the minimal regions implicated in oxidn. of each compd. were detd. The region required for oxidn. of phosphite to phosphate putatively encodes a binding-protein-dependent phosphite transporter, an NAD+-dependent phosphite dehydrogenase, and a transcriptional activator of the lysR family. The region required for oxidn. of hypophosphite to phosphite putatively encodes a binding-protein-dependent hypophosphite transporter and an a-ketoglutarate-dependent hypophosphite dioxygenase. The finding of genes dedicated to oxidn. of reduced P compds. provides further evidence that a redox cycle for P may be important in the metab. of this essential, and often growth-limiting, nutrient.
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    82. 82
      Polyviou, D.; Hitchcock, A.; Baylay, A. J.; Moore, C. M.; Bibby, T. S. Phosphite Utilization by the Globally Important Marine Diazotroph Trichodesmium. Environ. Microbiol. Rep. 2015, 7 (6), 824– 830,  DOI: 10.1111/1758-2229.12308
      82
      Phosphite utilization by the globally important marine diazotroph Trichodesmium
      Polyviou, Despo; Hitchcock, Andrew; Baylay, Alison J.; Moore, C. Mark; Bibby, Thomas S.
      Environmental Microbiology Reports (2015), 7 (6), 824-830CODEN: EMRNAG; ISSN:1758-2229. (Wiley-Blackwell)
      Summary : Species belonging to the filamentous cyanobacterial genus Trichodesmium are responsible for a significant fraction of oceanic nitrogen fixation. The availability of phosphorus (P) likely constrains the growth of Trichodesmium in certain regions of the ocean. Moreover, Trichodesmium species have recently been shown to play a role in an emerging oceanic phosphorus redox cycle, further highlighting the key role these microbes play in many biogeochem. processes in the contemporary ocean. Here, we show that Trichodesmium erythraeum IMS101 can grow on the reduced inorg. compd. phosphite as its sole source of P. The components responsible for phosphite utilization are identified through heterologous expression of the T. erythraeum IMS101 Tery_0365-0368 genes, encoding a putative ATP (ATP)-binding cassette transporter and NAD (NAD)-dependent dehydrogenase, in the model cyanobacteria Synechocystis sp. PCC6803. We demonstrate that only combined expression of both the transporter and the dehydrogenase enables Synechocystis to utilize phosphite, confirming the function of Tery_0365-0367 as a phosphite uptake system (PtxABC) and Tery_0368 as a phosphite dehydrogenase (PtxD). Our findings suggest that reported uptake of phosphite by Trichodesmium consortia in the field likely reflects an active biol. process by Trichodesmium. These results highlight the diversity of phosphorus sources available to Trichodesmium in a resource-limited ocean.
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    83. 83
      Feingersch, R.; Philosof, A.; Mejuch, T.; Glaser, F.; Alalouf, O.; Shoham, Y.; Béjà, O. Potential for Phosphite and Phosphonate Utilization by Prochlorococcus. ISME J. 2012, 6 (4), 827– 834,  DOI: 10.1038/ismej.2011.149
      83
      Potential for phosphite and phosphonate utilization by Prochlorococcus
      Feingersch, Roi; Philosof, Alon; Mejuch, Tom; Glaser, Fabian; Alalouf, Onit; Shoham, Yuval; Beja, Oded
      ISME Journal (2012), 6 (4), 827-834CODEN: IJSOCF; ISSN:1751-7362. (Nature Publishing Group)
      Phosphonates (Pn) are diverse org. phosphorus (P) compds. contg. C-P bonds and comprise up to 25% of the high-mol. wt. dissolved org. P pool in the open ocean. Pn bioavailability was suggested to influence markedly bacterial primary prodn. in low-P areas. Using metagenomic data from the Global Ocean Sampling expedition, we show that the main potential microbial contributor in Pn utilization in oceanic surface water is the globally important marine primary producer Prochlorococcus. Moreover, a no. of Prochlorococcus strains contain two distinct putative Pn uptake operons coding for ABC-type Pn transporters. On the basis of microcalorimetric measurements, we find that each of the two different putative Pn-binding protein (PhnD) homologs transcribed from these operons possesses different Pn- as well as inorg. phosphite-binding specificities. Our results suggest that Prochlorococcus adapt to low-P environments by increasing the no. of Pn transporters with different specificities towards phosphite and different Pns.
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    84. 84
      Bisson, C.; Adams, N. B. P.; Stevenson, B.; Brindley, A. A.; Polyviou, D.; Bibby, T. S.; Baker, P. J.; Hunter, C. N.; Hitchcock, A. The Molecular Basis of Phosphite and Hypophosphite Recognition by ABC-Transporters. Nat. Commun. 2017, 8 (1), 1– 12,  DOI: 10.1038/s41467-017-01226-8
      84
      The molecular basis of phosphite and hypophosphite recognition by ABC-transporters
      Bisson, Claudine; Adams, Nathan B. P.; Stevenson, Ben; Brindley, Amanda A.; Polyviou, Despo; Bibby, Thomas S.; Baker, Patrick J.; Hunter, C. Neil; Hitchcock, Andrew
      Nature Communications (2017), 8 (1), 1-13CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Inorg. phosphate is the major bioavailable form of the essential nutrient phosphorus. However, the concn. of phosphate in most natural habitats is low enough to limit microbial growth. Under phosphate-depleted conditions some bacteria utilize phosphite and hypophosphite as alternative sources of phosphorus, but the mol. basis of reduced phosphorus acquisition from the environment is not fully understood. Here, we present crystal structures and ligand binding affinities of periplasmic binding proteins from bacterial phosphite and hypophosphite ATP-binding cassette transporters. We reveal that phosphite and hypophosphite specificity results from a combination of steric selection and the presence of a P-H...π interaction between the ligand and a conserved arom. residue in the ligand-binding pocket. The characterization of high affinity and specific transporters has implications for the marine phosphorus redox cycle, and might aid the use of phosphite as an alternative phosphorus source in biotechnol., industrial and agricultural applications.
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    85. 85
      Jumper, J. Highly Accurate Protein Structure Prediction with AlphaFold. Nature 2021, 596 (7873), 583– 589,  DOI: 10.1038/s41586-021-03819-2
      85
      Highly accurate protein structure prediction with AlphaFold
      Jumper, John; Evans, Richard; Pritzel, Alexander; Green, Tim; Figurnov, Michael; Ronneberger, Olaf; Tunyasuvunakool, Kathryn; Bates, Russ; Zidek, Augustin; Potapenko, Anna; Bridgland, Alex; Meyer, Clemens; Kohl, Simon A. A.; Ballard, Andrew J.; Cowie, Andrew; Romera-Paredes, Bernardino; Nikolov, Stanislav; Jain, Rishub; Adler, Jonas; Back, Trevor; Petersen, Stig; Reiman, David; Clancy, Ellen; Zielinski, Michal; Steinegger, Martin; Pacholska, Michalina; Berghammer, Tamas; Bodenstein, Sebastian; Silver, David; Vinyals, Oriol; Senior, Andrew W.; Kavukcuoglu, Koray; Kohli, Pushmeet; Hassabis, Demis
      Nature (London, United Kingdom) (2021), 596 (7873), 583-589CODEN: NATUAS; ISSN:0028-0836. (Nature Portfolio)
      Proteins are essential to life, and understanding their structure can facilitate a mechanistic understanding of their function. Through an enormous exptl. effort, the structures of around 100,000 unique proteins have been detd., but this represents a small fraction of the billions of known protein sequences. Structural coverage is bottlenecked by the months to years of painstaking effort required to det. a single protein structure. Accurate computational approaches are needed to address this gap and to enable large-scale structural bioinformatics. Predicting the three-dimensional structure that a protein will adopt based solely on its amino acid sequence-the structure prediction component of the 'protein folding problem'-has been an important open research problem for more than 50 years. Despite recent progress, existing methods fall far short of at. accuracy, esp. when no homologous structure is available. Here we provide the first computational method that can regularly predict protein structures with at. accuracy even in cases in which no similar structure is known. We validated an entirely redesigned version of our neural network-based model, AlphaFold, in the challenging 14th Crit. Assessment of protein Structure Prediction (CASP14), demonstrating accuracy competitive with exptl. structures in a majority of cases and greatly outperforming other methods. Underpinning the latest version of AlphaFold is a novel machine learning approach that incorporates phys. and biol. knowledge about protein structure, leveraging multi-sequence alignments, into the design of the deep learning algorithm.
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    86. 86
      Gupta, R.; Laxman, S. Cycles, Sources, and Sinks: Conceptualizing How Phosphate Balance Modulates Carbon Flux Using Yeast Metabolic Networks. Elife 2021, 10, e63341,  DOI: 10.7554/eLife.63341
      86
      Cycles, sources, and sinks: conceptualizing how phosphate balance modulates carbon flux using yeast metabolic networks
      Gupta, Ritu; Laxman, Sunil
      eLife (2021), 10 (), e63341CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
      Phosphates are ubiquitous mols. that enable crit. intracellular biochem. reactions. Therefore, cells have elaborate responses to phosphate limitation. Our understanding of long-term transcriptional responses to phosphate limitation is extensive. Contrastingly, a systemslevel perspective presenting unifying biochem. concepts to interpret how phosphate balance is critically coupled to (and controls) metabolic information flow is missing. To conceptualize such processes, utilizing yeast metabolic networks we categorize phosphates utilized in metab. into cycles, sources and sinks. Through this, we identify metabolic reactions leading to putative phosphate sources or sinks. With this conceptualization, we illustrate how mass action driven flux towards sources and sinks enable cells to manage phosphate availability during transient/ immediate phosphate limitations. We thereby identify how intracellular phosphate availability will predictably alter specific nodes in carbon metab., and det. signature cellular metabolic states. Finally, we identify a need to understand intracellular phosphate pools, in order to address mechanisms of phosphate regulation and restoration.
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    87. 87
      Simeonova, D. D.; Wilson, M. M.; Metcalf, W. W.; Schink, B. Identification and Heterologous Expression of Genes Involved in Anaerobic Dissimilatory Phosphite Oxidation by Desulfotignum Phosphitoxidans. J. Bacteriol. 2010, 192 (19), 5237– 5244,  DOI: 10.1128/JB.00541-10
      87
      Identification and heterologous expression of genes involved in anaerobic dissimilatory phosphite oxidation by Desulfotignum phosphitoxidans
      Simeonova, Diliana Dancheva; Wilson, Marlena Marie; Metcalf, William W.; Schink, Bernhard
      Journal of Bacteriology (2010), 192 (19), 5237-5244CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      Desulfotignum phosphitoxidans is a strictly anaerobic, Gram-neg. bacterium that utilizes phosphite as the sole electron source for homoacetogenic CO2 redn. or sulfate redn. A genomic library of D. phosphitoxidans, constructed using the fosmid vector pJK050, was screened for clones harboring the genes involved in phosphite oxidn. via PCR using primers developed based on the amino acid sequences of phosphite-induced proteins. Sequence anal. of two pos. clones revealed a putative operon of seven genes predicted to be involved in phosphite oxidn. Four of these genes (ptxD-ptdFCG) were cloned and heterologously expressed in Desulfotignum balticum, a related strain that cannot use phosphite as either an electron donor or as a phosphorus source. The ptxD-ptdFCG gene cluster was sufficient to confer phosphite uptake and oxidn. ability to the D. balticum host strain but did not allow use of phosphite as an electron donor for chemolithotrophic growth. Phosphite oxidn. activity was measured in cell exts. of D. balticum transconjugants, suggesting that all genes required for phosphite oxidn. were cloned. Genes of the phosphite gene cluster were assigned putative functions on the basis of sequence anal. and enzyme assays.
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    88. 88
      van Veen, H. W.; Abee, T.; Kortstee, G. J. J.; Konings, W. N.; Zehnder, A. J. B. Translocation of Metal Phosphate via the Phosphate Inorganic Transport System of Escherichia coli. Biochemistry 1994, 33 (7), 1766– 1770,  DOI: 10.1021/bi00173a020
      88
      Translocation of Metal phosphate via the Phosphate Inorganic Transport System of Escherichia coli
      van Veen, Hendrik W.; Abee, Tjakko; Kortstee, Gerard J. J.; Konings, Wil N.; Zehnder, Alexander J. B.
      Biochemistry (1994), 33 (7), 1766-70CODEN: BICHAW; ISSN:0006-2960.
      Pi transport via the phosphate inorg. transport system (Pit) of E. coli was studied in natural and artificial membranes. Pi uptake via Pit is dependent on the presence of divalent cations, like Mg2+, Ca2+, Co2+, or Mn2+, which form a sol., neutral metal phosphate (MeHPO4) complex. Pi-dependent uptake of Mg2+ and Ca2+, equimolar cotransport of Pi and Ca2+, and inhibition by Mg2+ of Ca2+ uptake in the presence of Pi, but not of Pi uptake in the presence of Ca2+, indicate that a metal phosphate complex is the transported solute. Metal phosphate is transported in symport with H+ with a mechanistic stoichiometry of 1. Pit mediates efflux and homologous exchange of metal phosphate, but not heterologous metal phosphate exchange with Pi, glycerol-3P, or glucose-6P. The metal phosphate efflux rate increased with pH, whereas the rate of metal phosphate exchange was essentially pH independent. Metal phosphate uptake was inhibited at low internal pH. Efflux was inhibited by a proton motive force (interior neg. and alk.), whereas exchange was inhibited by the membrane potential only. These results have been evaluated in terms of ordered binding and dissocn. of metal phosphate and protons on the outer and inner surface of the cytoplasmic membrane.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXht12hsbg%253D&md5=295d6b1bd59160f7dc247092ddfa7029
    89. 89
      Pedersen, B. P.; Kumar, H.; Waight, A. B.; Risenmay, A. J.; Roe-Zurz, Z.; Chau, B. H.; Schlessinger, A.; Bonomi, M.; Harries, W.; Sali, A.; Johri, A. K.; Stroud, R. M. Crystal Structure of a Eukaryotic Phosphate Transporter. Nature 2013, 496 (7446), 533– 536,  DOI: 10.1038/nature12042
      89
      Crystal structure of a eukaryotic phosphate transporter
      Pedersen, Bjorn P.; Kumar, Hemant; Waight, Andrew B.; Risenmay, Aaron J.; Roe-Zurz, Zygy; Chau, Bryant H.; Schlessinger, Avner; Bonomi, Massimiliano; Harries, William; Sali, Andrej; Johri, Atul K.; Stroud, Robert M.
      Nature (London, United Kingdom) (2013), 496 (7446), 533-536CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Phosphate is crucial for structural and metabolic needs, including nucleotide and lipid synthesis, signalling and chem. energy storage. Proton-coupled transporters of the major facilitator superfamily (MFS) are essential for phosphate uptake in plants and fungi, and also have a function in sensing external phosphate levels as transceptors. Here we report the 2.9 Å structure of a fungal (Piriformospora indica) high-affinity phosphate transporter, PiPT, in an inward-facing occluded state, with bound phosphate visible in the membrane-buried binding site. The structure indicates both proton and phosphate exit pathways and suggests a modified asym. 'rocker-switch' mechanism of phosphate transport. PiPT is related to several human transporter families, most notably the org. cation and anion transporters of the solute carrier family (SLC22), which are implicated in cancer-drug resistance. We modelled representative cation and anion SLC22 transporters based on the PiPT structure to surmise the structural basis for substrate binding and charge selectivity in this important family. The PiPT structure demonstrates and expands on principles of substrate transport by the MFS transporters and illuminates principles of phosphate uptake in particular.
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    90. 90
      de Pinto, V.; Tommasino, M.; Palmieri, F.; Kadenbach, B. Purification of the Active Mitochondrial Phosphate Carrier by Affinity Chromatography with an Organomercurial Agarose Column. FEBS Lett. 1982, 148 (1), 103– 106,  DOI: 10.1016/0014-5793(82)81252-0
      90
      Purification of the active mitochondrial phosphate carrier by affinity chromatography with an organomercurial agarose column
      De Pinto, V.; Tommasino, M.; Palmieri, F.; Kadenbach, B.
      FEBS Letters (1982), 148 (1), 103-6CODEN: FEBLAL; ISSN:0014-5793.
      Active phosphate carrier was purified by affinity chromatog. from a hydroxylapatite eluate of Triton X 100-solubilized mitochondria. Affinity chromatog. was performed with Affi-Gel 501 as stationary phase and with a mercaptoethanol gradient in the mobile phase. One main protein band was obsd. after high-resoln. SDS gel electrophoresis of this prepn.
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    91. 91
      Motomura, K.; Hirota, R.; Ohnaka, N.; Okada, M.; Ikeda, T.; Morohoshi, T.; Ohtake, H.; Kuroda, A. Overproduction of YjbB Reduces the Level of Polyphosphate in Escherichia coli: A Hypothetical Role of YjbB in Phosphate Export and Polyphosphate Accumulation. FEMS Microbiol. Lett. 2011, 320 (1), 25– 32,  DOI: 10.1111/j.1574-6968.2011.02285.x
      91
      Overproduction of YjbB reduces the level of polyphosphate in Escherichia coli: a hypothetical role of YjbB in phosphate export and polyphosphate accumulation
      Motomura, Kei; Hirota, Ryuichi; Ohnaka, Nobuteru; Okada, Mai; Ikeda, Takeshi; Morohoshi, Tomohiro; Ohtake, Hisao; Kuroda, Akio
      FEMS Microbiology Letters (2011), 320 (1), 25-32CODEN: FMLED7; ISSN:0378-1097. (Wiley-Blackwell)
      Intracellular phosphate (Pi) is normally maintained at a fairly const. concn. in Escherichia coli, mainly by Pi transport systems and by the "phosphate balance" between Pi and polyphosphate (polyP). We have reported previously that excess uptake of Pi in a phoU mutant results in elevated levels of polyP. Here, we found that the elevated levels of polyP in the mutant could be reduced by the overprodn. of YjbB, whose N-terminal half contains Na+/Pi cotransporter domains. The rate of Pi export increased when the YjbB overproducer grew on a medium contg. glycerol-3-phosphate. These results strongly suggested that YjbB reduced the elevated levels of polyP in the phoU mutant by exporting intracellular excess Pi.
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    92. 92
      Hamburger, D.; Rezzonico, E.; MacDonald-Comber Petetot, J.; Somerville, C.; Poirier, Y. Identification and Characterization of the Arabidopsis PHO1 Gene Involved in Phosphate Loading to the Xylem. Plant Cell 2002, 14 (4), 889– 902,  DOI: 10.1105/tpc.000745
      92
      Identification and characterization of the Arabidopsis PHO1 gene involved in phosphate loading to the xylem
      Hamburger, Dirk; Rezzonico, Enea; Petetot, Jean MacDonald-Comber; Somerville, Chris; Poirier, Yves
      Plant Cell (2002), 14 (4), 889-902CODEN: PLCEEW; ISSN:1040-4651. (American Society of Plant Biologists)
      The Arabidopsis mutant pho1 is deficient in the transfer of Pi from root epidermal and cortical cells to the xylem. The PHO1 gene was identified by a map-based cloning strategy. The N-terminal half of PHO1 is mainly hydrophilic, whereas the C-terminal half has six potential membrane-spanning domains. PHO1 shows no homol. with any characterized solute transporter, including the family of H+-Pi cotransporters identified in plants and fungi. PHO1 shows highest homol. with the Rcm1 mammalian receptor for xenotropic murine leukemia retroviruses and with the Saccharomyces cerevisiae Syg1 protein involved in the mating pheromone signal transduction pathway. PHO1 is expressed predominantly in the roots and is upregulated weakly under Pi stress. Studies with PHO1 promoter-β-glucuronidase constructs reveal predominant expression of the PHO1 promoter in the stelar cells of the root and the lower part of the hypocotyl. There also is β-glucuronidase staining of endodermal cells that are adjacent to the protoxylem vessels. The Arabidopsis genome contains 10 addnl. genes showing homol. with PHO1. Thus, PHO1 defines a novel class of proteins involved in ion transport in plants.
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    93. 93
      Palmieri, F.; Indiveri, C.; Bisaccia, F.; Krämer, R. Functional Properties of Purified and Reconstituted Mitochondrial Metabolite Carriers. J. Bioenerg. Biomembr. 1993, 25 (5), 525– 535,  DOI: 10.1007/BF01108409
      93
      Functional properties of purified and reconstituted mitochondrial metabolite carriers
      Palmieri, F.; Indiveri, C.; Bisaccia, F.; Kraemer, R.
      Journal of Bioenergetics and Biomembranes (1993), 25 (5), 525-35CODEN: JBBID4; ISSN:0145-479X.
      A review with ∼60 refs. Eight mitochondrial carrier proteins were solubilized and purified in the authors' labs. using variations of a general procedure based on hydroxyapatite and Celite chromatog. The mol. mass of all the carriers ranges between 28 and 34 kDa on SDS-PAGE. The purified carrier proteins were reconstituted into liposomes mainly by using a method of detergent removal by hydrophobic chromatog. on polystyrene beads. The various carriers were identified in the reconstituted state by their kinetic properties. A complete set of basic kinetic data including substrate specificity, affinity, interaction with inhibitors, and activation energy was obtained. These data closely resemble those of intact mitochondria, as far as they are available from the intact organelle. Mainly on the basis of kinetic data, the asym. orientation of most of the reconstituted carrier proteins were established. Several of their functional properties are significantly affected by the type of phospholipids used for reconstitution.
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    94. 94
      Seddon, A. M.; Curnow, P.; Booth, P. J. Membrane Proteins, Lipids and Detergents: Not Just a Soap Opera. Biochim. Biophys. Acta - Biomembr. 2004, 1666 (1–2), 105– 117,  DOI: 10.1016/j.bbamem.2004.04.011
      94
      Membrane proteins, lipids and detergents: not just a soap opera
      Seddon, Annela M.; Curnow, Paul; Booth, Paula J.
      Biochimica et Biophysica Acta, Biomembranes (2004), 1666 (1-2), 105-117CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
      A review. Studying membrane proteins represents a major challenge in protein biochem., with one of the major difficulties being the problems encountered when working outside the natural lipid environment. In vitro studies such as crystn. are reliant on the successful solubilization or reconstitution of membrane proteins, which generally involves the careful selection of solubilizing detergents and mixed lipid/detergent systems. This review will conc. on the methods currently available for efficient reconstitution and solubilization of membrane proteins through the use of detergent micelles, mixed lipid/detergent micelles and bicelles or liposomes. We focus on the relevant mol. properties of the detergents and lipids that aid understanding of these processes. A significant barrier to membrane protein research is retaining the stability and function of the protein during solubilization, reconstitution and crystn. We highlight some of the lessons learnt from studies of membrane protein folding in vitro and give an overview of the role that lipids can play in stabilizing the proteins.
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    95. 95
      Junge, F.; Schneider, B.; Reckel, S.; Schwarz, D.; Dötsch, V.; Bernhard, F. Large-Scale Production of Functional Membrane Proteins. Cell. Mol. Life Sci. 2008, 65 (11), 1729– 1755,  DOI: 10.1007/s00018-008-8067-5
      95
      Large-scale production of functional membrane proteins
      Junge, F.; Schneider, B.; Reckel, S.; Schwarz, D.; Doetsch, V.; Bernhard, F.
      Cellular and Molecular Life Sciences (2008), 65 (11), 1729-1755CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Verlag)
      A review. The prepn. of sufficient amts. of high-quality samples is still the major bottleneck for the characterization of membrane proteins by in vitro approaches. The hydrophobic nature, the requirement for complicated transport and modification pathways, and the often obsd. neg. effects on membrane properties are intrinsic features of membrane proteins that frequently cause significant problems in overexpression studies. Establishing efficient protocols for the prodn. of functionally folded membrane proteins is therefore a challenging task, and numerous specific characteristics have to be considered. In addn., a variety of expression systems have been developed, and choice of appropriate techniques could strongly depend on the desired target membrane proteins as well as on their intended applications. The prodn. of membrane proteins is a highly dynamic field and new or modified approaches are frequently emerging. The review will give an overview of currently established processes for the prodn. of functionally folded membrane proteins.
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    96. 96
      Amati, A. M.; Graf, S.; Deutschmann, S.; Dolder, N.; von Ballmoos, C. Current Problems and Future Avenues in Proteoliposome Research. Biochem. Soc. Trans. 2020, 48 (4), 1473– 1492,  DOI: 10.1042/BST20190966
      96
      Current problems and future avenues in proteoliposome research
      Amati, Andrea Marco; Graf, Simone; Deutschmann, Sabina; Dolder, Nicolas; von Ballmoos, Christoph
      Biochemical Society Transactions (2020), 48 (4), 1473-1492CODEN: BCSTB5; ISSN:0300-5127. (Portland Press Ltd.)
      A review. Membrane proteins (MPs) are the gatekeepers between different biol. compartments sepd. by lipid bilayers. Being receptors, channels, transporters, or primary pumps, they fulfill a wide variety of cellular functions and their importance is reflected in the increasing no. of drugs that target MPs. Functional studies of MPs within a native cellular context, however, is difficult due to the innate complexity of the densely packed membranes. Over the past decades, detergent-based extn. and purifn. of MPs and their reconstitution into lipid mimetic systems has been a very powerful tool to simplify the exptl. system. In this review, we focus on proteoliposomes that have become an indispensable exptl. system for enzymes with a vectorial function, including many of the here described energy transducing MPs. We first address long standing questions on the difficulty of successful reconstitution and controlled orientation of MPs into liposomes. A special emphasis is given on coreconstitution of several MPs into the same bilayer. Second, we discuss recent progress in the development of fluorescent dyes that offer sensitive detection with high temporal resoln. Finally, we briefly cover the use of giant unilamellar vesicles for the investigation of complex enzymic cascades, a very promising exptl. tool considering our increasing knowledge of the interplay of different cellular components.
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    97. 97
      Warnecke, T.; Gill, R. T. Organic Acid Toxicity, Tolerance, and Production in Escherichia coli Biorefining Applications. Microb. Cell Fact. 2005, 4 (1), 1– 8,  DOI: 10.1186/1475-2859-4-25
      There is no corresponding record for this reference.
    98. 98
      Voskuhl, L.; Brusilova, D.; Brauer, V. S.; Meckenstock, R. U. Inhibition of Sulfate-Reducing Bacteria with Formate. FEMS Microbiol. Ecol. 2022, 98 (1), 1– 10,  DOI: 10.1093/femsec/fiac003
      There is no corresponding record for this reference.
    99. 99
      Gabba, M.; Frallicciardi, J.; van ’t Klooster, J.; Henderson, R.; Syga, Ł.; Mans, R.; van Maris, A. J. A.; Poolman, B. Weak Acid Permeation in Synthetic Lipid Vesicles and Across the Yeast Plasma Membrane. Biophys. J. 2020, 118 (2), 422– 434,  DOI: 10.1016/j.bpj.2019.11.3384
      99
      Weak Acid Permeation in Synthetic Lipid Vesicles and Across the Yeast Plasma Membrane
      Gabba, Matteo; Frallicciardi, Jacopo; van 't Klooster, Joury; Henderson, Ryan; Syga, Lukasz; Mans, Robert; van Maris, Antonius J. A.; Poolman, Bert
      Biophysical Journal (2020), 118 (2), 422-434CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      The authors present a fluorescence-based approach for detn. of the permeability of small mols. across the membranes of lipid vesicles and living cells. With properly designed expts., the method allows the authors to assess the membrane phys. properties both in vitro and in vivo. The permeability of weak acids increases in the order of benzoic > acetic > formic > lactic, both in synthetic lipid vesicles and the plasma membrane of Saccharomyces cerevisiae, but the permeability is much lower in yeast (one to two orders of magnitude). A relation between the mol. permeability and the satn. of the lipid acyl chain (i.e., lipid packing) in the synthetic lipid vesicles. were obsd. By analyzing wild-type yeast and a manifold knockout strain lacking all putative lactic acid transporters, the yeast plasma membrane is impermeable to lactic acid on timescales up to ∼2.5 h.
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    100. 100
      Amao, Y. Formate Dehydrogenase for CO2 Utilization and Its Application. J. CO2 Util. 2018, 26, 623– 641,  DOI: 10.1016/j.jcou.2018.06.022
      100
      Formate dehydrogenase for CO2 utilization and its application
      Amao, Yutaka
      Journal of CO2 Utilization (2018), 26 (), 623-641CODEN: JCUOAJ; ISSN:2212-9839. (Elsevier Ltd.)
      Carbon dioxide, CO2 redn. and utilization for org. compds. synthesis are the potential technologies in environmental science and technol. In order to establish efficient CO2 utilization technologies, an effective catalyst for CO2 redn. and utilization is necessary. Among various catalysts, the biocatalyst is one of promising catalysts because it has excellent selectivity for the reaction and substrate. In this review, focusing on biocatalyst "formate dehydrogenase FDH" catalyzing CO2 redn. to formic acid, representative examples of properties, types, structure of active-site of FDH and, reaction mechanism of formic acid oxidn. and CO2 redn. with FDH are outlined. A genetic engineering modified FDH and FDH immobilized various support for improving CO2 redn. catalytic activity also are introduced. Moreover, chem. and electrochem. system of CO2 redn. to formic acid with FDH, aq. homogenous system of visible-light driven CO2 redn. to formic acid with FDH and device for visible-light driven CO2 redn. to formic acid with FDH are also introduced as an application of FDH.
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    101. 101
      Maia, L. B.; Moura, I.; Moura, J. J. G. Molybdenum and Tungsten-Containing Formate Dehydrogenases: Aiming to Inspire a Catalyst for Carbon Dioxide Utilization. Inorg. Chim. Acta 2017, 455, 350– 363,  DOI: 10.1016/j.ica.2016.07.010
      101
      Molybdenum and tungsten-containing formate dehydrogenases: Aiming to inspire a catalyst for carbon dioxide utilization
      Maia, Luisa B.; Moura, Isabel; Moura, Jose J. G.
      Inorganica Chimica Acta (2017), 455 (Part_2), 350-363CODEN: ICHAA3; ISSN:0020-1693. (Elsevier B.V.)
      A review concerning the use Mo- and W-contg. formate dehydrogenase (FDH) enzymes as a model to understand mechanistic strategies and key chem. features needed to reduce CO2 to formate, highlighting current knowledge about the FDH structure, emphasizing active site features, reaction mechanism, and ability to reduce CO2 to formate, is given. The gathered information aims to inspire development of new efficient bio-catalysts for atm. CO2 utilization to produce energy and chem. feedstocks while reducing an important environmental pollutant. Topics covered include: the CO2 crisis; FDH enzymes (families, Mo- and W-contg. FDH, mechanistic strategies for FDH, new look at FDH catalysis); FDH-catalyzed CO2 redn.; and outlook.
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    102. 102
      Tishkov, V. I.; Matorin, A. D.; Rojkova, A. M.; Fedorchuk, V. V.; Savitsky, P. A.; Dementieva, L. A.; Lamzin, V. S.; Mezentzev, A. V.; Popov, V. O. Site-Directed Mutagenesis of the Formate Dehydrogenase Active Centre: Role of the His332-Gln313 Pair in Enzyme Catalysis. FEBS Lett. 1996, 390 (1), 104– 108,  DOI: 10.1016/0014-5793(96)00641-2
      102
      Site-directed mutagenesis of the formate dehydrogenase active center: role of the His332-Gln313 pair in enzyme catalysis
      Tishkov, Vladimir I.; Matorin, Andrey D.; Rojkova, Alexandra M.; Fedorchuk, Vladimir V.; Savitsky, Pavel A.; Dementieva, Larissa A.; Lamzin, Victor S.; Mezentzev, Alexander V.; Popov, Vladimir O.
      FEBS Letters (1996), 390 (1), 104-108CODEN: FEBLAL; ISSN:0014-5793. (Elsevier)
      Gln313 and His332 residues in the active center of NAD+-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from the bacterium Pseudomonas sp. 101 are conserved in all FDHs and are equiv. to the glutamate-histidine pair in active sites of D-specific 2-hydroxy acid dehydrogenases. Two mutants of formate dehydrogenase from Pseudomonas sp. 101, Gln313Glu and His332Phe, have been obtained and characterized. The Gln313Glu mutation shifts the pK of the group controlling formate binding from less than 5.5 in wild-type enzyme to 7.6 thus indicating that Gln313 is essential for the broad pH affinity profile towards substrate. His332Phe mutation leads to a complete loss of enzyme activity. The His332Phe mutant is still able to bind coenzyme but not substrate or analogs. The role of histidine in the active center of FDH is discussed. The protonation state of His332 is not crit. for catalysis but vital for substrate binding. A partial pos. charge on the histidine imidazole, required for substrate binding, is provided via tight H-bond to the Gln313 carboxamide.
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    103. 103
      Lamzin, V. S.; Dauter, Z.; Popov, V. O.; Harutyunyan, E. H.; Wilson, K. S. High Resolution Structures of Holo and Apo Formate Dehydrogenase. J. Mol. Biol. 1994, 236, 759– 785,  DOI: 10.1006/jmbi.1994.1188
      103
      High resolution structures of holo and apo formate dehydrogenase
      Lamzin, Victor S.; Dauter, Zbigniew; Popov, Vladimir O.; Harutyunyan, Emil H.; Wilson, Keith S.
      Journal of Molecular Biology (1994), 236 (3), 759-85CODEN: JMOBAK; ISSN:0022-2836.
      Three-dimensional crystal structures of holo (ternary complex enzyme-NAD-azide) and apo NAD-dependent dimeric formate dehydrogenase (FDH) from the methylotrophic bacterium Pseudomonas sp. 101 have been refined to R factors of 11.7% and 14.8% at 2.05 and 1.80 Å resoln., resp. The estd. root-mean-square error in at. coordinates is 0.11 Å for holo and 0.18 Å for apo. X-ray data were collected from single crystals using an imaging plate scanner and synchrotron radiation. In both crystal forms there is a dimer in the asym. unit. Both structures show essentially 2-fold mol. symmetry. NAD binding causes movement of the catalytic domain and ordering of the C terminus, where a new helix appears. This completes formation of the enzyme active center in holo FDH. NAD is bound in the cleft sepg. the domains and mainly interacts with residues from the co-enzyme binding domain. In apo FDH these residues are held in essentially the same conformation by water mols. occupying the NAD binding region. An azide mol. is located near the point of catalysis, the C4 atom of the nicotinamide moiety of NAD, and overlaps with the proposed formate binding site. There is an extensive channel running from the active site to the protein surface and this is supposed to be used by substrate to reach the active center after NAD has already bound. The structure of the active site and a hypothetical catalytic mechanism are discussed. Sequence homol. of FDH with other NAD-dependent formate dehydrogenases and some D-specific dehydrogenases is discussed on the basis of the FDH three-dimensional structure.
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    104. 104
      Hoelsch, K.; Sührer, I.; Heusel, M.; Weuster-Botz, D. Engineering of Formate Dehydrogenase: Synergistic Effect of Mutations Affecting Cofactor Specificity and Chemical Stability. Appl. Microbiol. Biotechnol. 2013, 97 (6), 2473– 2481,  DOI: 10.1007/s00253-012-4142-9
      104
      Engineering of formate dehydrogenase: synergistic effect of mutations affecting cofactor specificity and chemical stability
      Hoelsch, Kathrin; Suehrer, Ilka; Heusel, Moritz; Weuster-Botz, Dirk
      Applied Microbiology and Biotechnology (2013), 97 (6), 2473-2481CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      Formate dehydrogenases (FDHs) are frequently used for the regeneration of cofactors in biotransformations employing NAD(P)H-dependent oxidoreductases. Major drawbacks of most native FDHs are their strong preference for NAD+ and their low operational stability in the presence of reactive org. compds. such as α-haloketones. In this study, the FDH from Mycobacterium vaccae N10 (MycFDH) was engineered in order to obtain an enzyme that is not only capable of regenerating NADPH but also stable toward the α-haloketone Et 4-chloroacetoacetate (ECAA). To change the cofactor specificity, amino acids in the conserved NAD+ binding motif were mutated. Among these mutants, MycFDH A198G/D221Q had the highest catalytic efficiency (kcat/K m) with NADP+. The addnl. replacement of two cysteines (C145S/C255V) not only conferred a high resistance to ECAA but also enhanced the catalytic efficiency 6-fold. The resulting quadruple mutant MycFDH C145S/A198G/D221Q/C255V had a specific activity of 4.00 ± 0.13 U mg-1 and a K of 0.147 ± 0.020 mM at 30 °C, pH 7. The A198G replacement had a major impact on the kinetic consts. of the enzyme. The corresponding triple mutant, MycFDH C145S/D221Q/C255V, showed the highest specific activity reported to date for a NADP+-accepting FDH (vmax, 10.25 ± 1.63 U mg-1). However, the half-satn. const. for NADP+ (Km, 0.92 ± 0.10 mM) was about one order of magnitude higher than the one of the quadruple mutant. Depending on the reaction setup, both novel MycFDH variants could be useful for the prodn. of the chiral synthon Et (S)-4-chloro-3-hydroxybutyrate [(S)-ECHB] by asym. redn. of ECAA with NADPH-dependent ketoreductases.
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    105. 105
      Calzadiaz-Ramirez, L.; Calvó-Tusell, C.; Stoffel, G. M. M.; Lindner, S. N.; Osuna, S.; Erb, T. J.; Garcia-Borràs, M.; Bar-Even, A.; Acevedo-Rocha, C. G. In Vivo Selection for Formate Dehydrogenases with High Efficiency and Specificity toward NADP+. ACS Catal. 2020, 10 (14), 7512– 7525,  DOI: 10.1021/acscatal.0c01487
      105
      In vivo selection for formate dehydrogenases with high efficiency and specificity toward NADP+
      Calzadiaz-Ramirez, Liliana; Calvo-Tusell, Carla; Stoffel, Gabriele M. M.; Lindner, Steffen N.; Osuna, Silvia; Erb, Tobias J.; Garcia-Borras, Marc; Bar-Even, Arren; Acevedo-Rocha, Carlos G.
      ACS Catalysis (2020), 10 (14), 7512-7525CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
      The efficient regeneration of cofactors is vital for the establishment of biocatalytic processes. Formate is an ideal electron donor for cofactor regeneration due to its general availability, low redn. potential, and benign byproduct (CO2). However, formate dehydrogenases (FDHs) are usually specific to NAD+, such that NADPH regeneration with formate is challenging. Previous studies reported naturally occurring FDHs or engineered FDHs that accept NADP+, but these enzymes show low kinetic efficiencies and specificities. Here, we harness the power of natural selection to engineer FDH variants to simultaneously optimize three properties: kinetic efficiency with NADP+, specificity toward NADP+, and affinity toward formate. By simultaneously mutating multiple residues of FDH from Pseudomonas sp. 101, which exhibits practically no activity toward NADP+, we generate a library of >106 variants. We introduce this library into an E. coli strain that cannot produce NADPH. By selecting for growth with formate as the sole NADPH source, we isolate several enzyme variants that support efficient NADPH regeneration. We find that the kinetically superior enzyme variant, harboring five mutations, has 5-fold higher efficiency and 14-fold higher specificity in comparison to the best enzyme previously engineered, while retaining high affinity toward formate. By using mol. dynamics simulations, we reveal the contribution of each mutation to the superior kinetics of this variant. We further det. how nonadditive epistatic effects improve multiple parameters simultaneously. Our work demonstrates the capacity of in vivo selection to identify highly proficient enzyme variants carrying multiple mutations which would be almost impossible to find using conventional screening methods.
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    106. 106
      Partipilo, M.; Whittaker, J.J.; Pontillo, N.; Coenradij, J.; Herrmann, A.; Guskov, A.; Slotboom, D. J. Biochemical and Structural Insight into the Chemical Resistance and Cofactor Specificity of the Formate Dehydrogenase from Starkeya Novella. FEBS J.
      There is no corresponding record for this reference.
    107. 107
      Endeward, V.; Al-Samir, S.; Itel, F.; Gros, G. How Does Carbon Dioxide Permeate Cell Membranes? A Discussion of Concepts, Results and Methods. Front. Physiol. 2014, 4, 382,  DOI: 10.3389/fphys.2013.00382
      107
      How does carbon dioxide permeate cell membranes? A discussion of concepts, results and methods
      Endeward Volker; Al-Samir Samer; Gros Gerolf; Itel Fabian
      Frontiers in physiology (2014), 4 (), 382 ISSN:1664-042X.
      We review briefly how the thinking about the permeation of gases, especially CO2, across cell and artificial lipid membranes has evolved during the last 100 years. We then describe how the recent finding of a drastic effect of cholesterol on CO2 permeability of both biological and artificial membranes fundamentally alters the long-standing idea that CO2-as well as other gases-permeates all membranes with great ease. This requires revision of the widely accepted paradigm that membranes never offer a serious diffusion resistance to CO2 or other gases. Earlier observations of "CO2-impermeable membranes" can now be explained by the high cholesterol content of some membranes. Thus, cholesterol is a membrane component that nature can use to adapt membrane CO2 permeability to the functional needs of the cell. Since cholesterol serves many other cellular functions, it cannot be reduced indefinitely. We show, however, that cells that possess a high metabolic rate and/or a high rate of O2 and CO2 exchange, do require very high CO2 permeabilities that may not be achievable merely by reduction of membrane cholesterol. The article then discusses the alternative possibility of raising the CO2 permeability of a membrane by incorporating protein CO2 channels. The highly controversial issue of gas and CO2 channels is systematically and critically reviewed. It is concluded that a majority of the results considered to be reliable, is in favor of the concept of existence and functional relevance of protein gas channels. The effect of intracellular carbonic anhydrase, which has recently been proposed as an alternative mechanism to a membrane CO2 channel, is analysed quantitatively and the idea considered untenable. After a brief review of the knowledge on permeation of O2 and NO through membranes, we present a summary of the (18)O method used to measure the CO2 permeability of membranes and discuss quantitatively critical questions that may be addressed to this method.
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    108. 108
      Lubitz, W.; Ogata, H.; Rudiger, O.; Reijerse, E. Hydrogenases. Chem. Rev. 2014, 114 (8), 4081– 4148,  DOI: 10.1021/cr4005814
      108
      Hydrogenases
      Lubitz, Wolfgang; Ogata, Hideaki; Ruediger, Olaf; Reijerse, Edward
      Chemical Reviews (Washington, DC, United States) (2014), 114 (8), 4081-4148CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. The current state of knowledge on hydrogenases, esp. recent advances made in understanding the detailed structure and function of these important enzymes. The authors provide an overview of important previous achievements with the main focus on [NiFe] and [FeFe] hydrogenases, and in part also on [Fe] hydrogenases. Recent progress on biomimetic model systems for hydrogenases and devices using hydrogenases both in fuel cells and for H2 prodn. are presented with emphasis on functional aspects. The great progress made in synthesizing model systems for hydrogenases that are functionally active is promising for the future employment of such catalysts in hydrogen technologies.
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    109. 109
      Lauterbach, L.; Lenz, O.; Vincent, K. A. H2-Driven Cofactor Regeneration with NAD(P)+-Reducing Hydrogenases. FEBS J. 2013, 280 (13), 3058– 3068,  DOI: 10.1111/febs.12245
      109
      H2-driven cofactor regeneration with NAD(P)+-reducing hydrogenases
      Lauterbach, Lars; Lenz, Oliver; Vincent, Kylie A.
      FEBS Journal (2013), 280 (13), 3058-3068CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
      A review. A large no. of industrially relevant enzymes depend upon nicotinamide cofactors, which are too expensive to be added in stoichiometric amts. Existing NAD(P)H-recycling systems suffer from low activity, or the generation of side products. H2-driven cofactor regeneration has the advantage of 100% atom efficiency and the use of H2 as a cheap reducing agent, in a world where sustainable energy carriers are increasingly attractive. The state of development of H2-driven cofactor-recycling systems and examples of their integration with enzyme reactions are summarized in this article. The O2-tolerant NAD+-reducing hydrogenase from Ralstonia eutropha is a particularly attractive candidate for this approach, and we therefore discuss its catalytic properties that are relevant for tech. applications.
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    110. 110
      Vandamme, P.; Coenye, T. Taxonomy of the Genus Cupriavidus: A Tale of Lost and Found. Int. J. Syst. Evol. Microbiol. 2004, 54 (6), 2285– 2289,  DOI: 10.1099/ijs.0.63247-0
      110
      Taxonomy of the genus Cupriavidus: a tale of lost and found
      Vandamme Peter; Coenye Tom
      International journal of systematic and evolutionary microbiology (2004), 54 (Pt 6), 2285-2289 ISSN:1466-5026.
      DNA-DNA hybridization experiments and an evaluation of phenotypic characteristics, DNA base ratios and 16S rRNA gene sequences demonstrated that Wautersia eutropha (Davies 1969) Vaneechoutte et al. 2004, the type species of the genus Wautersia, is a later synonym of Cupriavidus necator Makkar and Casida 1987, the type species of the genus Cupriavidus. In conformity with Rules 15, 17, 23a and 37a(1) of the International Code of Nomenclature of Bacteria, the genus name Cupriavidus has priority over the genus name Wautersia, and all other members of the genus Wautersia are reclassified into Cupriavidus as Cupriavidus basilensis comb. nov. (type strain LMG 18990(T)=DSM 11853(T)), Cupriavidus campinensis comb. nov. (type strain LMG 19282(T)=CCUG 44526(T)), Cupriavidus gilardii comb. nov. (type strain LMG 5886(T)=CCUG 38401(T)), Cupriavidus metallidurans comb. nov. (type strain LMG 1195(T)=DSM 2839(T)), Cupriavidus oxalaticus comb. nov. (type strain LMG 2235(T)=CCUG 2086(T)=DSM 1105(T)), Cupriavidus pauculus comb. nov. (type strain LMG 3244(T)=CCUG 12507(T)), Cupriavidus respiraculi comb. nov. (type strain LMG 21510(T)=CCUG 46809(T)) and Cupriavidus taiwanensis comb. nov. (type strain LMG 19424(T)=CCUG 44338(T)).
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    111. 111
      Shomura, Y.; Taketa, M.; Nakashima, H.; Tai, H.; Nakagawa, H.; Ikeda, Y.; Ishii, M.; Igarashi, Y.; Nishihara, H.; Yoon, K. S.; Ogo, S.; Hirota, S.; Higuchi, Y. Structural Basis of the Redox Switches in the NAD+-Reducing Soluble [NiFe]-Hydrogenase. Science. 2017, 357 (6354), 928– 932,  DOI: 10.1126/science.aan4497
      111
      Structural basis of the redox switches in the NAD+-reducing soluble [NiFe]-hydrogenase
      Shomura, Y.; Taketa, M.; Nakashima, H.; Tai, H.; Nakagawa, H.; Ikeda, Y.; Ishii, M.; Igarashi, Y.; Nishihara, H.; Yoon, K.-S.; Ogo, S.; Hirota, S.; Higuchi, Y.
      Science (Washington, DC, United States) (2017), 357 (6354), 928-932CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      NAD-reducing sol. [NiFe]-hydrogenase (SH) is phylogenetically related to NADH:quinone oxidoreductase (complex I), but the geometrical arrangements of the subunits and Fe-S clusters are unclear. Here, we describe the crystal structures of SH of Hydrogenophilus thermoluteolus in the oxidized and reduced states. The cluster arrangement was similar to that of complex I, but the subunits orientation was not, which supported the hypothesis that subunits evolved as prebuilt modules. The oxidized active site included a 6-coordinate Ni, which was unprecedented for hydrogenases, whose coordination geometry would prevent O2 from approaching. In the reduced state showing the normal active site structure without a physiol. electron acceptor, the FMN cofactor was dissocd., which may be caused by the oxidn. state change of nearby Fe-S clusters and may suppress the prodn. of reactive oxygen species.
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    112. 112
      Kulka-Peschke, C. J.; Schulz, A. C.; Lorent, C.; Rippers, Y.; Wahlefeld, S.; Preissler, J.; Schulz, C.; Wiemann, C.; Bernitzky, C. C. M.; Karafoulidi-Retsou, C.; Wrathall, S. L. D.; Procacci, B.; Matsuura, H.; Greetham, G. M.; Teutloff, C.; Lauterbach, L.; Higuchi, Y.; Ishii, M.; Hunt, N. T.; Lenz, O.; Zebger, I.; Horch, M. Reversible Glutamate Coordination to High-Valent Nickel Protects the Active Site of a [NiFe] Hydrogenase from Oxygen. J. Am. Chem. Soc. 2022, 144 (37), 17022– 17032,  DOI: 10.1021/jacs.2c06400
      112
      Reversible Glutamate Coordination to High-Valent Nickel Protects the Active Site of a [NiFe] Hydrogenase from Oxygen
      Kulka-Peschke, Catharina J.; Schulz, Anne-Christine; Lorent, Christian; Rippers, Yvonne; Wahlefeld, Stefan; Preissler, Janina; Schulz, Claudia; Wiemann, Charlotte; Bernitzky, Cornelius C. M.; Karafoulidi-Retsou, Chara; Wrathall, Solomon L. D.; Procacci, Barbara; Matsuura, Hiroaki; Greetham, Gregory M.; Teutloff, Christian; Lauterbach, Lars; Higuchi, Yoshiki; Ishii, Masaharu; Hunt, Neil T.; Lenz, Oliver; Zebger, Ingo; Horch, Marius
      Journal of the American Chemical Society (2022), 144 (37), 17022-17032CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      NAD+-reducing [NiFe] hydrogenases are valuable biocatalysts for H2-based energy conversion and the regeneration of nucleotide cofactors. While most hydrogenases are sensitive toward O2 and elevated temps., the sol. NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus (HtSH) is O2-tolerant and thermostable. Thus, it represents a promising candidate for biotechnol. applications. Here, we have investigated the catalytic activity and active-site structure of native HtSH and variants in which a glutamate residue in the active-site cavity was replaced by glutamine, alanine, and aspartate. Our biochem., spectroscopic, and theor. studies reveal that at least two active-site states of oxidized HtSH feature an unusual architecture in which the glutamate acts as a terminal ligand of the active-site nickel. This observation demonstrates that crystallog. obsd. glutamate coordination represents a native feature of the enzyme. One of these states is diamagnetic and characterized by a very high stretching frequency of an iron-bound active-site CO ligand. Supported by d.-functional-theory calcns., we identify this state as a high-valent species with a biol. unprecedented formal Ni(IV) ground state. Detailed insights into its structure and dynamics were obtained by ultrafast and two-dimensional IR spectroscopy, demonstrating that it represents a conformationally strained state with unusual bond properties. Our data further show that this state is selectively and reversibly formed under oxic conditions, esp. upon rapid exposure to high O2 levels. We conclude that the kinetically controlled formation of this six-coordinate high-valent state represents a specific and precisely orchestrated stereoelectronic response toward O2 that could protect the enzyme from oxidative damage.
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    113. 113
      Preissler, J.; Reeve, H. A.; Zhu, T.; Nicholson, J.; Urata, K.; Lauterbach, L.; Wong, L. L.; Vincent, K. A.; Lenz, O. Dihydrogen-Driven NADPH Recycling in Imine Reduction and P450-Catalyzed Oxidations Mediated by an Engineered O2-Tolerant Hydrogenase. ChemCatChem. 2020, 12 (19), 4853– 4861,  DOI: 10.1002/cctc.202000763
      113
      Dihydrogen-Driven NADPH Recycling in Imine Reduction and P450-Catalyzed Oxidations Mediated by an Engineered O2-Tolerant Hydrogenase
      Preissler, Janina; Reeve, Holly A.; Zhu, Tianze; Nicholson, Jake; Urata, Kouji; Lauterbach, Lars; Wong, Luet L.; Vincent, Kylie A.; Lenz, Oliver
      ChemCatChem (2020), 12 (19), 4853-4861CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The O2-tolerant NAD+-reducing hydrogenase (SH) from Ralstonia eutropha (Cupriavidus necator) has already been applied in vitro and in vivo for H2-driven NADH recycling in coupled enzymic reactions with various NADH-dependent oxidoreductases. To expand the scope for application in NADPH-dependent biocatalysis, we introduced changes in the NAD+-binding pocket of the enzyme by rational mutagenesis, and generated a variant with significantly higher affinity for NADP+ than for the natural substrate NAD+, while retaining native O2-tolerance. The applicability of the SH variant in H2-driven NADPH supply was demonstrated by the full conversion of 2-methyl-1-pyrroline into a single enantiomer of 2-methylpyrrolidine catalyzed by a stereoselective imine reductase. In an even more challenging reaction, the SH supported a cytochrome P 450 monooxygenase for the oxidn. of octane under safe H2/O2 mixts. Thus, the re-designed SH represents a versatile platform for atom-efficient, H2-driven cofactor recycling in biotransformations involving NADPH-dependent oxidoreductases.
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    114. 114
      Cammack, R.; Fernandez, V. M.; Hatchikian, E. C. [5] Nickel-Iron Hydrogenase. Methods Enzym. 1994, 243 (1989), 43– 68,  DOI: 10.1016/0076-6879(94)43007-1
      There is no corresponding record for this reference.
    115. 115
      Yagi, T.; Honya, M.; Tamiya, N. Purification and Properties of Hydrogenases of Different Origins. Biochim. Biophys. Acta (BBA)-Bioenergetics 1968, 153 (3), 699– 705,  DOI: 10.1016/0005-2728(68)90197-7
      There is no corresponding record for this reference.
    116. 116
      Spencer, P.; Bown, K. J.; Scawen, M. D.; Atkinson, T.; Gore, M. G. Isolation and Characterisation of the Glycerol Dehydrogenase from Bacillus Stearothermophilus. Biochim. Biophys. Acta (BBA)/Protein Struct. Mol. 1989, 994 (3), 270– 279,  DOI: 10.1016/0167-4838(89)90304-X
      There is no corresponding record for this reference.
    117. 117
      Iyer, P. V.; Ananthanarayan, L. Enzyme Stability and Stabilization-Aqueous and Non-Aqueous Environment. Process Biochem. 2008, 43 (10), 1019– 1032,  DOI: 10.1016/j.procbio.2008.06.004
      117
      Enzyme stability and stabilization-Aqueous and non-aqueous environment
      Iyer, Padma V.; Ananthanarayan, Laxmi
      Process Biochemistry (Amsterdam, Netherlands) (2008), 43 (10), 1019-1032CODEN: PBCHE5; ISSN:1359-5113. (Elsevier B.V.)
      A review. Enzyme stabilization has notable importance due to increasing no. of enzyme applications. Stabilization of enzymes in order to realize their full potential as catalysts is discussed in the present review. An overview of the denaturation mechanisms in aq. and non-aq. environment is given. Further various methods of enzyme stabilization with respect to their use in aq. and non-aq. environment have been given. Using thermophilic enzymes as the ref. point, a review of stabilization using various approaches like protein engineering, chem. modifications of enzymes and immobilization has been attempted. Finally, it has been stressed that, for selection of a suitable stabilization approach the intended use and possible interactions between the stabilizer-enzyme have to be taken into consideration.
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    118. 118
      Łapińska, U.; Kahveci, Z.; Irwin, N. A. T.; Milner, D. S.; Santoro, A. E.; Richards, T. A.; Pagliara, S. Membrane Permeability Differentiation at the Lipid Divide. PLoS biology 2023, 21 (4), e3002048,  DOI: 10.1371/journal.pbio.3002048
      There is no corresponding record for this reference.
    119. 119
      Rigoulet, M.; Bouchez, C. L.; Paumard, P.; Ransac, S.; Cuvellier, S.; Duvezin-Caubet, S.; Mazat, J. P.; Devin, A. Cell Energy Metabolism: An Update. Biochim. Biophys. Acta - Bioenerg. 2020, 1861 (11), 148276,  DOI: 10.1016/j.bbabio.2020.148276
      119
      Cell energy metabolism: An update
      Rigoulet, M.; Bouchez, C. L.; Paumard, P.; Ransac, S.; Cuvellier, S.; Duvezin-Caubet, S.; Mazat, J. P.; Devin, A.
      Biochimica et Biophysica Acta, Bioenergetics (2020), 1861 (11), 148276CODEN: BBBEB4; ISSN:0005-2728. (Elsevier B.V.)
      A review. In living cells, growth is the result of coupling between substrate catabolism and multiple metabolic processes that take place during net biomass formation and maintenance processes. During growth, both ATP/ADP and NADH/NAD+ mols. play a key role. Cell energy metab. hence refers to metabolic pathways involved in ATP synthesis linked to NADH turnover. Two main pathways are thus involved in cell energy metab.: glycolysis/fermn. and oxidative phosphorylation. Glycolysis and mitochondrial oxidative phosphorylation are intertwined through thermodn. and kinetic constraints that are reviewed herein. Further, our current knowledge of short-term and long term regulation of cell energy metab. will be reviewed using examples such as the Crabtree and the Warburg effect.
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    120. 120
      Pollak, N.; Dölle, C.; Ziegler, M. The Power to Reduce: Pyridine Nucleotides - Small Molecules with a Multitude of Functions. Biochem. J. 2007, 402 (2), 205– 218,  DOI: 10.1042/BJ20061638
      120
      The power to reduce: pyridine nucleotides - small molecules with a multitude of functions
      Pollak, Nadine; Doelle, Christian; Ziegler, Mathias
      Biochemical Journal (2007), 402 (2), 205-218CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
      A review. The pyridine nucleotides NAD and NADP play vital roles in metabolic conversions as signal transducers and in cellular defense systems. Both coenzymes participate as electron carriers in energy transduction and biosynthetic processes. Their oxidized forms, NAD+ and NADP+, have been identified as important elements of regulatory pathways. In particular, NAD+ serves as a substrate for ADP-ribosylation reactions and for the Sir2 family of NAD+-dependent protein deacetylases as well as a precursor of the calcium mobilizing mol. cADPr (cyclic ADP-ribose). The conversions of NADP+ into the 2'-phosphorylated form of cADPr or to its nicotinic acid deriv., NAADP, also result in the formation of potent intracellular calcium-signalling agents. Perhaps, the most crit. function of NADP is in the maintenance of a pool of reducing equiv. which is essential to counteract oxidative damage and for other detoxifying reactions. It is well known that the NADPH/NADP+ ratio is usually kept high, in favor of the reduced form. Research within the past few years has revealed important insights into how the NADPH pool is generated and maintained in different subcellular compartments. Moreover, tremendous progress in the mol. characterization of NAD kinases has established these enzymes as vital factors for cell survival. In the present review, we summarize recent advances in the understanding of the biosynthesis and signalling functions of NAD(P) and highlight the new insights into the mol. mechanisms of NADPH generation and their roles in cell physiol.
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    121. 121
      Tishkov, V. I.; Popov, V. O. Protein Engineering of Formate Dehydrogenase. Biomol. Eng. 2006, 23 (2–3), 89– 110,  DOI: 10.1016/j.bioeng.2006.02.003
      121
      Protein engineering of formate dehydrogenase
      Tishkov, Vladimir I.; Popov, Vladimir O.
      Biomolecular Engineering (2006), 23 (2-3), 89-110CODEN: BIENFV; ISSN:1389-0344. (Elsevier B.V.)
      A review. NAD-dependent formate dehydrogenase (FDH; EC 1.2.1.2) is one of the best enzymes for the purpose of NADH regeneration in dehydrogenase-based synthesis of optically active compds. Low operational stability and high prodn. cost of native FDHs limit their application in com. prodn. of chiral compds. Here, the authors summarize the results on engineering of bacterial and yeast FDHs aimed at improving their chem. and thermal stability, catalytic activity, switch in coenzyme specificity from NAD to NADP, and overexpression in Escherichia coli cells.
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    122. 122
      Bouzon, M.; Döring, V.; Dubois, I.; Berger, A.; Stoffel, G. M. M.; Ramirez, L. C.; Meyer, S. N.; Fouré, M.; Roche, D.; Perret, A.; Erb, T. J.; Bar-Even, A.; Lindner, S. N. Change in Cofactor Specificity of Oxidoreductases by Adaptive Evolution of an Escherichia coli Nadph-Auxotrophic Strain. MBio 2021, 12 (4), e00329-21,  DOI: 10.1128/mBio.00329-21
      There is no corresponding record for this reference.
    123. 123
      Chánique, A. M.; Parra, L. P. Protein Engineering for Nicotinamide Coenzyme Specificity in Oxidoreductases: Attempts and Challenges. Front. Microbiol. 2018,  DOI: 10.3389/fmicb.2018.00194
      There is no corresponding record for this reference.
    124. 124
      Rydström, J.; Hoek, J. B.; Ernster, L. 2 Nicotinamide Nucleotide Transhydrogenases. The Enzymes 1976, 13, 51– 88,  DOI: 10.1016/S1874-6047(08)60240-1
      There is no corresponding record for this reference.
    125. 125
      Hoek, J. B.; Rydstrom, J. Physiological Roles of Nicotinamide Nucleotide Transhydrogenase. Biochem. J. 1988, 254 (1), 1– 10,  DOI: 10.1042/bj2540001
      125
      Physiological roles of nicotinamide nucleotide transhydrogenase
      Hoek, Jan B.; Rydstroem, Jan
      Biochemical Journal (1988), 254 (1), 1-10CODEN: BIJOAK; ISSN:0264-6021.
      A review, with 93 refs., on the kinetic and thermodn. characteristics of the title transhydrogenase and its functions, e.g. as a redox buffer in the supply of reducing equiv., and protection of the mitochondria NADP redox state, as well as hormone effects on the enzyme.
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    126. 126
      Van de Stadt, R. J.; Nieuwenhuis, F. J. R. M.; Van dam, K. On the Reversibility of the Energy-Linked Transhydrogenase. BBA - Bioenerg. 1971, 234 (1), 173– 176,  DOI: 10.1016/0005-2728(71)90143-5
      There is no corresponding record for this reference.
    127. 127
      Pedersen, A.; Karlsson, G. B.; Rydström, J. Proton-Translocating Transhydrogenase: An Update of Unsolved and Controversial Issues. J. Bioenerg. Biomembr. 2008, 40 (5), 463– 473,  DOI: 10.1007/s10863-008-9170-x
      127
      Proton-translocating transhydrogenase: an update of unsolved and controversial issues
      Pedersen, Anders; Karlsson, Goeran B.; Rydstroem, Jan
      Journal of Bioenergetics and Biomembranes (2008), 40 (5), 463-473CODEN: JBBID4; ISSN:0145-479X. (Springer)
      A review. H+-translocating transhydrogenases, reducing NADP to NADH through hydride transfer, are membrane enzymes utilizing the electrochem. proton gradient for NADPH generation. These enzymes have important physiol. roles in the maintenance of e.g., reduced glutathione, relevant for essentially all cell types. Following x-ray crystallog. and structural resoln. of the sol. substrate-binding domains, mechanistic aspects of the hydride transfer reaction are beginning to be resolved. However, the structure of the intact enzyme is still unknown. Key questions regarding the coupling mechanism, i.e., the mechanism of proton translocation, are addressed using the sep. expressed substrate-binding domains. Important aspects are therefore which functions and properties of mainly the sol. NADP(H)-binding domain (but also the NAD(H)-binding domain) are relevant for proton translocation, how the sol. domains communicate with the membrane domain, and the mechanism of proton translocation through the membrane domain.
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    128. 128
      Leung, J. H.; Schurig-Briccio, L. A.; Yamaguchi, M.; Moeller, A.; Speir, J. A.; Gennis, R. B.; Stout, C. D. Division of Labor in Transhydrogenase by Alternating Proton Translocation and Hydride Transfer. Science. 2015, 347 (6218), 178– 181,  DOI: 10.1126/science.1260451
      128
      Division of labor in transhydrogenase by alternating proton translocation and hydride transfer
      Leung, Josephine H.; Schurig-Briccio, Lici A.; Yamaguchi, Mutsuo; Moeller, Arne; Speir, Jeffrey A.; Gennis, Robert B.; Stout, Charles D.
      Science (Washington, DC, United States) (2015), 347 (6218), 178-181CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      NADPH/NADP+ (the reduced form of NADP+/NADP) homeostasis is crit. for countering oxidative stress in cells. Nicotinamide nucleotide transhydrogenase (TH), a membrane enzyme present in both bacteria and mitochondria, couples the proton motive force (PMF) to the generation of NADPH. We present the 2.8 Å crystal structure of the transmembrane proton channel domain of TH from Thermus thermophilus and the 6.9 Å crystal structure of the entire enzyme (holo-TH). The membrane domain crystd. as a sym. dimer, with each protomer contg. a putative proton channel. The holo-TH is a highly asym. dimer with the NADP(H)-binding domain (dIII) in two different orientations. This unusual arrangement suggests a catalytic mechanism in which the two copies of dIII alternatively function in proton translocation and hydride transfer.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitFemuw%253D%253D&md5=20710c99c6b97abf58c9fab995f271b4
    129. 129
      Kampjut, D.; Sazanov, L. A. Structure and Mechanism of Mitochondrial Proton-Translocating Transhydrogenase. Nature 2019, 573 (7773), 291– 295,  DOI: 10.1038/s41586-019-1519-2
      129
      Structure and mechanism of mitochondrial proton-translocating transhydrogenase
      Kampjut, Domen; Sazanov, Leonid A.
      Nature (London, United Kingdom) (2019), 573 (7773), 291-295CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      Proton-translocating transhydrogenase (also known as nicotinamide nucleotide transhydrogenase (NNT)) is found in the plasma membranes of bacteria and the inner mitochondrial membranes of eukaryotes. NNT catalyzes the transfer of a hydride between NADH and NADP+, coupled to the translocation of one proton across the membrane. Its main physiol. function is the generation of NADPH, which is a substrate in anabolic reactions and a regulator of oxidative status; however, NNT may also fine-tune the Krebs cycle1,2. NNT deficiency causes familial glucocorticoid deficiency in humans and metabolic abnormalities in mice, similar to those obsd. in type II diabetes3,4. The catalytic mechanism of NNT has been proposed to involve a rotation of around 180° of the entire NADP(H)-binding domain that alternately participates in hydride transfer and proton-channel gating. However, owing to the lack of high-resoln. structures of intact NNT, the details of this process remain unclear5,6. Here we present the cryo-electron microscopy structure of intact mammalian NNT in different conformational states. We show how the NADP(H)-binding domain opens the proton channel to the opposite sides of the membrane, and we provide structures of these two states. We also describe the catalytically important interfaces and linkers between the membrane and the sol. domains and their roles in nucleotide exchange. These structures enable us to propose a revised mechanism for a coupling process in NNT that is consistent with a large body of previous biochem. work. Our results are relevant to the development of currently unavailable NNT inhibitors, which may have therapeutic potential in ischemia reperfusion injury, metabolic syndrome and some cancers7-9.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1Oht7jK&md5=0df4ee25fddb7d2f3d8a518405612280
    130. 130
      Enander, K.; Rydström, J. Energy-Linked Nicotinamide Nucleotide Transhydrogenase. Kinetics and Regulation of Purified and Reconstituted Transhydrogenase from Beef Heart Mitochondria. J. Biol. Chem. 1982, 257 (24), 14760– 14766,  DOI: 10.1016/S0021-9258(18)33345-3
      130
      Energy-linked nicotinamide nucleotide transhydrogenase. Kinetics and regulation of purified and reconstituted transhydrogenase from beef heart mitochondria
      Enander, Kirsten; Rydstroem, Jan
      Journal of Biological Chemistry (1982), 257 (24), 14760-6CODEN: JBCHA3; ISSN:0021-9258.
      Purified and reconstituted nicotinamide nucleotide transhydrogenase (I) from bovine heart mitochondria was investigated with respect to kinetic and regulatory properties in uncoupled and coupled liposomes. Double reciprocal plots of initial velocities for the redn. of NADPH vs. substrate concns. were convergent and intersecting on or close to the abscissa, indicating a ternary complex mechanism. The effect of site-specific inhibitors indicated that the order of addn. of the substrates to I was random. Reconstituted I uncoupled by FCCP revealed kinetic properties that were indicative of energization, i.e., an increased and decreased affinity for NADP and NAD, resp., suggesting that reconstituted I is maintained in an activated conformation. An increased extent of coupling caused a progressively increasing change in the same direction. Apparently, the uncoupler-dependent enhancement of the rate of redn. of NAD by NADPH is due to a decreased Km for NAD. Reconstituted I catalyzed a transhydrogenation between NADH and 3-acetylpyridine adenine dinucleotide (oxidized) in the presence of NADPH. Reconstituted I also catalyzed the redn. of thio-NADP by NADPH in the presence of NADH. Both reactions occurred indirectly through the generation of NADP and NAD, resp., and not directly through a reduced I intermediate. A H+-pump mechanism is proposed for I which involves a dimeric form of I where the 2 subunits alternate in H+ pumping.
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    131. 131
      Graf, S. S.; Hong, S.; Müller, P.; Gennis, R.; von Ballmoos, C. Energy Transfer between the Nicotinamide Nucleotide Transhydrogenase and ATP Synthase of Escherichia coli. Sci. Rep. 2021, 11 (1), 1– 12,  DOI: 10.1038/s41598-021-00651-6
      There is no corresponding record for this reference.
    132. 132
      Sauer, U.; Canonaco, F.; Heri, S.; Perrenoud, A.; Fischer, E. The Soluble and Membrane-Bound Transhydrogenases UdhA and PntAB Have Divergent Functions in NADPH Metabolism of Escherichia coli. J. Biol. Chem. 2004, 279 (8), 6613– 6619,  DOI: 10.1074/jbc.M311657200
      132
      The soluble and membrane-bound transhydrogenases UdhA and PntAB have divergent functions in NADPH metabolism of Escherichia coli
      Sauer, Uwe; Canonaco, Fabrizio; Heri, Sylvia; Perrenoud, Annik; Fischer, Eliane
      Journal of Biological Chemistry (2004), 279 (8), 6613-6619CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Pentose phosphate pathway and isocitrate dehydrogenase are generally considered to be the major sources of the anabolic reductant NADPH. As one of very few microbes, Escherichia coli contains two transhydrogenase isoforms with unknown physiol. function that could potentially transfer electrons directly from NADH to NADP+ and vice versa. Using defined mutants and metabolic flux anal., we identified the proton-translocating transhydrogenase PntAB as a major source of NADPH in E. coli. During std. aerobic batch growth on glucose, 35-45% of the NADPH that is required for biosynthesis was produced via PntAB, whereas pentose phosphate pathway and isocitrate dehydrogenase contributed 35-45% and 20-25%, resp. The energy-independent transhydrogenase UdhA, in contrast, was essential for growth under metabolic conditions with excess NADPH formation, i.e. growth on acetate or in a phosphoglucose isomerase mutant that catabolized glucose through the pentose phosphate pathway. Thus, both isoforms have divergent physiol. functions: energy-dependent redn. of NADP+ with NADH by PntAB and reoxidn. of NADPH by UdhA. Expression appeared to be modulated by the redox state of cellular metab., because genetic and environmental manipulations that increased or decreased NADPH formation down-regulated pntA or udhA transcription, resp. The two transhydrogenase isoforms provide E. coli primary metab. with an extraordinary flexibility to cope with varying catabolic and anabolic demands, which raises two general questions: why do only a few bacteria contain both isoforms, and how do other organisms manage NADPH metab.
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    133. 133
      Boonstra, B.; Bjorklund, L.; French, C. E.; Wainwright, I.; Bruce, N. C. Cloning of the Sth Gene from Azotobacter Vinelandii and Construction of Chimeric Soluble Pyridine Nucleotide Transhydrogenases. FEMS Microbiol. Lett. 2000, 191 (1), 87– 93,  DOI: 10.1111/j.1574-6968.2000.tb09323.x
      133
      Cloning of the sth gene from Azotobacter vinelandii and construction of chimeric soluble pyridine nucleotide transhydrogenases
      Boonstra, B.; Bjorklund, L.; French, C. E.; Wainwright, I.; Bruce, N. C.
      FEMS Microbiology Letters (2000), 191 (1), 87-93CODEN: FMLED7; ISSN:0378-1097. (Elsevier Science B.V.)
      The gene encoding the sol. pyridine nucleotide transhydrogenase (STH) of Azotobacter vinelandii was cloned and sequenced. This is the third sth gene identified and further defines a new subfamily within the flavoprotein disulfide oxidoreductases. The three STHs identified all lack one of the redox active cysteines that are characteristic for this large family of enzymes, and instead they contain a conserved threonine residue at this position. The recombinant A. vinelandii enzyme was purified to homogeneity and shown to form filamentous structures different from those of Pseudomonas fluorescens and Escherichia coli STH. Chimeric STHs were constructed which showed that the C-terminal region is important for polymer formation. The A. vinelandii STH contg. the C-terminal region of P. fluorescens or E. coli STH showed structures resembling those of the STH contributing the C-terminal portion of the protein.
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    134. 134
      Voordouw, G.; Veeger, C.; Breemen, J. F. L.; Bruggen, E. F. J. Structure Of Pyridine Nucleotide Transhydrogenase From Azotobacter Vinelandii. Eur. J. Biochem. 1979, 98 (2), 447– 454,  DOI: 10.1111/j.1432-1033.1979.tb13205.x
      134
      Structure of pyridine nucleotide transhydrogenase from azotobacter vinelandii
      Voordouw, Gerrit; Veeger, Cees; Van Breemen, Jan F. L.; Van Bruggen, Ernst F. J.
      European Journal of Biochemistry (1979), 98 (2), 447-54CODEN: EJBCAI; ISSN:0014-2956.
      Pyridine nucleotide transhydrogenase (I) of A. vinelandii purified by affinity chromatog. consisted of a mixt. of polydisperse rods at neutral pH. No other structures were seen by electron microscopy. A high pH (8.5-9.0) the rods depolymd. Complete depolymn. was achieved in 0.1M Tris-Cl pH 9.0. Depolymd. I has a mol. wt. of 421,000 (sedimentation equil.), sedimentation coeff. of 15 S, and its Stokes' radius is 7 nm. Since gel electrophoresis in the presence of Na dodecyl sulfate show that I consists of a single polypeptide chain of mol. wt. 54 × 103, it follows that depolymd. I has an octameric quaternary structure. This octamer may serve as the functional monomeric unit (unimer) from which the polymeric form of I is constructed. Gel filtration and sucrose gradient centrifugation studies of cell-free exts. show the unimer to be the predominant active species.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1MXlvVyltLg%253D&md5=720ebb78208fe220dfbaa11ff5ef4c88
    135. 135
      Partipilo, M.; Yang, G.; Mascotti, M. L.; Wijma, H. J.; Slotboom, D. J.; Fraaije, M. W. A Conserved Sequence Motif in the Escherichia coli Soluble FAD-Containing Pyridine Nucleotide Transhydrogenase Is Important for Reaction Efficiency. J. Biol. Chem. 2022, 298 (9), 102304,  DOI: 10.1016/j.jbc.2022.102304
      135
      A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency
      Partipilo, Michele; Yang, Guang; Mascotti, Maria Laura; Wijma, Hein J.; Slotboom, Dirk Jan; Fraaije, Marco W.
      Journal of Biological Chemistry (2022), 298 (9), 102304CODEN: JBCHA3; ISSN:1083-351X. (Elsevier Inc.)
      Sol. pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equiv. between the two nicotinamide cofactors. The sol. transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equiv. toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochem. characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with mol. oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational anal. revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with mol. oxygen and the importance of the evolutionarily conserved sequence motif.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XisVKnt7zF&md5=a1e1679e9999a73b87a0a6d7013a5eac
    136. 136
      Cao, Z.; Song, P.; Xu, Q.; Su, R.; Zhu, G. Overexpression and Biochemical Characterization of Soluble Pyridine Nucleotide Transhydrogenase from Escherichia coli. FEMS Microbiol. Lett. 2011, 320 (1), 9– 14,  DOI: 10.1111/j.1574-6968.2011.02287.x
      136
      Overexpression and biochemical characterization of soluble pyridine nucleotide transhydrogenase from Escherichia coli
      Cao, Zhengyu; Song, Ping; Xu, Qin; Su, Ruirui; Zhu, Guoping
      FEMS Microbiology Letters (2011), 320 (1), 9-14CODEN: FMLED7; ISSN:0378-1097. (Wiley-Blackwell)
      Sol. pyridine nucleotide transhydrogenase (STH) is an energy-independent flavoprotein that directly catalyzes hydride transfer between NAD(H) and NADP(H) to maintain homeostasis of these 2 redox cofactors. Here, the sth gene of E. coli was cloned and expressed as a fused protein (EcSTH). Purified EcSTH displayed maximal activity at 35° and pH 7.5. Heat-inactivation studies showed that EcSTH retained 50% activity after 5 h at 50°. The enzyme was stable at 4° for 25 days. The apparent Km values of EcSTH were 68.29 μM for NADPH and 133.2 μM for thio-NAD. The kcat/Km ratios showed that EcSTH had a 1.25-fold preference for NADPH over thio-NAD. Product inhibition studies showed that EcSTH activity was strongly inhibited by excess NADPH, but not by thio-NAD. EcSTH activity was enhanced by 2 mM adenine nucleotide and inhibited by divalent metal ions, including Mn2+, Co2+, Zn2+, Ni2+, and Cu2+. However, after preincubation for 30 min, most divalent metal ions had little effect on EcSTH activity, except Zn2+, Ni2+, and Cu2+. This enzymic anal. provides important basic knowledge for EcSTH utilization.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXnvFCnsb4%253D&md5=0d1ab36725bf568374069eb92db0f568
    137. 137
      Ighodaro, O. M.; Akinloye, O. A. First Line Defence Antioxidants-Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione Peroxidase (GPX): Their Fundamental Role in the Entire Antioxidant Defence Grid. Alexandria J. Med. 2018, 54 (4), 287– 293,  DOI: 10.1016/j.ajme.2017.09.001
      There is no corresponding record for this reference.
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